Molecular genetic mapping of dwarfing genes in oat

 Restriction fragment length polymorphism (RFLP) analysis provides a valuable tool for characterizing and understanding relationships among genes for useful traits in crop species, particularly in ones with complex genomes such as the hexaploid cultivated oat Avena sativa L. (2n=6x=42). Using Bulked Segregant Analysis (BSA) and F₂ RFLP linkage data, we mapped three dominant oat dwarfing loci to different regions of the oat genome. Dw6, in oat line OT207, is 3.3±1.3 cM from the Xumn145B locus, which has not been placed on the hexaploid oat linkage map. Dw7, in line NC2469-3, is 4.3±2.3 cM from Xcdo1437B and 33±4.1 cM from Xcdo708B. This places Dw7 to linkage group 22. Dw8, in the Japanese lines AV17/3/10 and AV18/2/4, mapped 4.9±2.2 cM from Xcdo1319A in an AV17/3/10בKanota’ F₂ population and 6.6±2.6 cM from it in an AV18/2/4בKanota’ population. This places Dw8 to linkage group 3. Aneuploid analysis of markers linked to the dwarfing genes located Dw6 on the smallest oat chromosome (chromosome 18) and Dw7 on the longest satellited chromosome (chromosome 19). The RFLP markers closely linked to the three dwarfing genes identify distinct regions of the oat genome that contribute to plant height and they should be useful in characterizing new genetic sources of dwarfness in oat. Received: 8 May 1997 / Accepted: 20 May 1997     

Detection Methanogens in Newly Settled Sediments from Xuanwu Lake in Nanjing, China

Sediments from Xuanwu Lake have been dredged in the past 3 years to improve the water quality, but methanogenesis should still exist in the newly settled sediment. Methane production, methanogens, and physiochemical parameters were detected in the surface sediments (0–5 cm) and/or vertical sediments (0–21 cm, segmented at interval of 3 cm). Methane flux at water–air interface varied among five detected sites. Principal component analysis showed that CH₄ flux, content of water and the concentration of total nitrogen (TN), CH₄ and organic matters (OM) weighed most heavily on the component I in surface sediments while different patterns were observed for vertical sediments. The copy number of the 16S rRNA gene for bacteria was lower in the surface sediment (0–6 cm) than that in deeper sediments (12–21 cm), while 16S rRNA genes of Archaea were almost evenly distributed in the vertical sediments. Representatives belonging to the orders Methanobacteriales, Methanomicrobiales, and Methanosarcinales were detected in all samples of the vertical sediments, except that no members of the Methanococcales were detected in the samples at 0–6 cm. The level of Methanobacteriales reached a highest density at 18.1 × 10⁴ copies g⁻¹ dry weight (dw) at 6–9 cm; for Methanosarcinales (76.89 × 10⁶ copies g⁻¹ dw) and Methanococcales (82.70 × 10³ copies g⁻¹ dw) at 12–15 cm, whereas for Methanomicrobiales (43.37 × 10⁶ copies g⁻¹ dw) at 9–12 cm. Methanosarcinaceae and Methanosaetaceae reached to their highest densities at 6–9 and 9–12 cm, respectively. These data provided useful information for better understanding the methanogenesis in the newly settled sediments of a recently dredged lake.     

Collinearity-based marker mining for the fine mapping of <Emphasis Type="Italic">Pm6</Emphasis>, a powdery mildew resistance gene in wheat

The genome sequences of rice (Oryza sativa L.) and Brachypodium distachyon and the comprehensive Triticeae EST (Expressed Sequence Tag) resources provide invaluable information for comparative genomics analysis. The powdery mildew resistance gene, Pm6, which was introgressed into common wheat from Triticum timopheevii, was previously mapped to the wheat chromosome bin of 2BL [fraction length (FL) 0.50–1.00] with limited DNA markers. In this study, we saturated the Pm6 locus in wheat using the collinearity-based markers by extensively exploiting these genomic resources. All wheat ESTs located in the bin 2BL FL 0.50–1.00 and their corresponding orthologous genes on rice chromosome 4 were firstly used to develop STS (Sequence Tagged Site) markers. Those identified markers that flanked the Pm6 locus were then used to identify the collinear regions in the genomes of rice and Brachypodium. Triticeae ESTs with orthologous genes in these collinear regions were further used to develop new conserved markers for the fine mapping of Pm6. Using two F₂ populations derived from crosses of IGVI-465 × Prins and IGVI-466 × Prins, we mapped a total of 29 markers to the Pm6 locus. Among them, 14 markers were co-segregated with Pm6 in the IGVI-466/Prins population. Comparative genome analysis showed that the collinear region of the 29 linked markers covers a ~5.6-Mb region in chromosome 5L of Brachypodium and a ~6.0-Mb region in chromosome 4L of rice. The marker order is conserved between rice and Brachypodium, but re-arrangements are present in wheat. Comparative mapping in the two populations showed that two conserved markers (CINAU123 and CINAU127) flanked the Pm6 locus, and an LRR-receptor-like protein kinase cluster was identified in the collinear regions of Brachypodium and rice. This putative resistance gene cluster provides a potential target site for further fine mapping and cloning of Pm6. Moreover, the newly developed conserved markers closely linked to Pm6 can be used for the marker-assisted selection (MAS) of Pm6 in wheat breeding programs.     

Development of three allele-specific codominant rice <Emphasis Type="Italic">Waxy</Emphasis> gene PCR markers suitable for marker-assisted selection of amylose content and paste viscosity

Four Waxy haplotypes, previously identified as each having a different combination of three single nucleotide polymorphisms (SNPs) in the Waxy gene, were highly correlated with apparent amylose content and pasting properties, which are important grain quality traits for predicting cooked rice (Oryza sativa L.) texture and processing properties (Chen et al. in J Cereal Sci 47:536–545, 2008a; Chen et al. in J Cereal Sci 48:781–788, 2008b). Three allele-specific PCR markers were developed to genotype the three aforementioned functional SNPs in a single PCR amplification. Each marker contained two allele-specific primers and one common primer. For each marker, the two allele-specific primers differed by one base at the 3′-end to provide discrimination of SNP alleles, and were labeled with unique fluorescence probes. An additional mismatched base, the third base from the 3′-end, was inserted in some allele-specific primers to increase selectivity. The amplification step of the PCR thermal cycling program was initially set for 20× touch-down cycles with the annealing temperature of the first cycle approximately 6°C above the thermal melting temperature of all three primers at a touch-down rate of −0.3°C per cycle, and followed by 25× regular thermal cycles with the annealing temperature at their thermal melting temperature. The allelic genotypes for each SNP were distinguished from each other by both their differential primer-allele fluorescences and their amplification product lengths. The simplicity of these assays makes it easy to utilize these markers as part of a marker-assisted selection strategy in rice breeding programs selecting for these important grain quality traits.     

Genetic mapping of the powdery mildew resistance gene Pm6 in wheat by RFLP analysis

Pm6 in bread wheat (Triticum aestivum L.), which was transferred from Triticum. timopheevii L., is a gene conferring resistance to the powdery mildew disease caused by Erysiphe graminis f. sp. tritici. Six near-isogenic lines ( NILs ) of Pm6 in a cultivar ’Prins’ background were analyzed to map this gene using restriction fragment length polymorphism (RFLP). Each of the six NILs possessed a T. timopheevii-derived segment, varying in length, and associated with powdery mildew resistance. Lines IGV1–465 (FAO163b/ 7*Prins) and IGV1–467 (Idaed 59B/7*Prins) had the shortest introgressed segments, which were detected only by DNA probes BCD135 and PSR934, respectively. The polymorphic loci detected by both probes were mapped to the long arm of chromosome 2B. Lines IGV1–458 (CI13250/7*Prins) and IGV1–456 (CI12559/8*Prins) contained the longest T. timopheevii segments involving both arms of donor chromosome 2G across the centromere. All these introgressed segments had an overlapping region flanked by the loci xpsr934 and xbcd135 on 2BL. Thus, Pm6 was located in this region since the powdery mildew resistance in all the NILs resulted from the introgressed fragments. Using the F₂ mapping population from a cross of IGV1–463 (PI170914/7*Prins)×Prins, Pm6 was shown to be closely linked to the loci xbcd135 and xbcd266 at a genetic distance of 1.6 cM and 4.8 cM, respectively. BCD135 was successfully used in detecting the presence of Pm6 in different genetic backgrounds. Received: 29 June 1999 / Accepted: 6 July 1999     

Kinetic models of photosystem II should accommodate the effect of donor side quenching on variable chlorophyll <Emphasis Type="Italic">a</Emphasis> fluorescence in the microseconds time range

Quantitative data on laser flash-induced variable fluorescence in the 100 ns to 1 ms time range (Belyaeva et al. in Photosynth Res 98:105–119, 2008) confirming those of others (Steffen et al. in Biochemistry 40:173–180, 2001, Biochemistry 44:3123–3132, 2005; Belyaeva et al. in Biophysics 51(6):976–990, 2006), need a substantial correction with respect to magnitude of the normalized variable fluorescence associated with single turnover-induced charge separation in RCs of PS II. Their data are conclusive with the involvement of donor side quenching, the release of which occurs with a rate constant in the range of tens of ms⁻¹, and presumed to be associated with reduction of Y_\textz ⁺ Y_{\text{z}}^{ + } by the OEC.     

A unique genome-wide association analysis in extended Utah high-risk pedigrees identifies a novel melanoma risk variant on chromosome arm 10q

Only two genome-wide association (GWA) screens have been published for melanoma (Nat Genet 47:920–925, 2009; Nat Genet 40:838–840, 2008). Using a unique approach, we performed a genome-wide association study in 156 related melanoma cases from 34 high-risk Utah pedigrees. Genome-wide association analysis was performed on nearly 500,000 markers; we compared cases to 2,150 genotypically matched samples from Illumina’s iControls database. We performed genome-wide association with EMMAX software, which is designed to account for population structure, including relatedness between cases. Three SNPs exceeded a genome-wide significance threshold of p < 5 × 10⁻⁸ on chromosome arm 10q25.1 (rs17119434, rs17119461, and rs17119490), where the most extreme p value was 7.21 × 10⁻¹². This study represents a new and unique approach to predisposition gene identification; and it is the first genome-wide association study performed in related cases in high-risk pedigrees. Our approach illustrates an example of using high-risk pedigrees for the identification of new melanoma predisposition variants.     

Identification of <Emphasis Type="Italic">Porphyra yezoensis</Emphasis> (Rhodophyta) meiosis by DNA quantification using confocal laser scanning microscopy

Conchospore germlings of Porphyra yezoensis were stained with a fluorescent dye for DNA and observed with confocal laser scanning microscopy (CLSM). Relative DNA values of the germling nuclei were obtained by measuring fluorescence intensities of nuclear regions of the optically sliced specimens, using the mean value of the smallest blade cells as a reference of the genomic n value. Such quantification revealed that the nuclear DNA amounts of the one-cell, two-cell, and four-cell-stage germlings are approximately 4 × n, 2 × n, and n ∼2 × n values respectively; these values agreed well with the expected ones from the hypothesis that meiosis corresponds to the first successive cell divisions after the conchospore germination. These results are consistent with a previous study on cytogenetic analysis of the chimaera blade formation (Ohme and Miura 1988, Plant Sci 57:135–140) and not consistent with a recent microscopic study (Wang et al. 2006, Phycol Res 54:201–207) which proposed that the first meiotic division occurs at the conchospore formation and the second division at the germination.     

<Emphasis Type="Italic">Dipcadi goaense</Emphasis> (Hyacinthaceae), a new species from the foothills of the Western Ghats,India

A new species of Dipcadi (Hyacinthaceae) that is allied to D. concanense (Dalzell) Baker but differs in its small flowers (13 – 18 mm long vs 35 – 47 mm long) and funnel shaped perianth tube (5 – 6  ×  5 – 6 mm vs 18 – 27  ×  4 – 5.5 mm) is described as D. goaense. The new species is apparently endemic, because it is known only from the type locality in Goa state of India. The type locality is at the foothills of Western Ghats and the habitat is a soil covered, lateritic, open area.     

Anthocyanin production in a callus line of <Emphasis Type="Italic">Panax sikkimensis</Emphasis> Ban

A root-derived callus line of Panax sikkimensis that stably accumulates anthocyanins was established by small cell aggregate selection method. The selected line showed a growth index of 221.36 and an anthocyanin content of 2.76 mg/g fw (7.076% dw) in 50–60 d of growth on a modified MS medium containing 4.5 μM 2,4-dichlorophenoxy acetic acid and 1.2 μM kinetin under 16-h light and 8-h dark photoperiodic conditions. Incubation under continuous light increased the growth index to 435.57 but led to a marginal dilution of anthocyanin content to 2.192 mg/g fw (6.928% dw). The purple-red pigment had absorption maximum at 528 nm. The selected callus line has shown sustained growth and productivity for more than 6 yr now. Interestingly, pigment accumulation in the selected line did not hinder the ginsenoside production in the callus tissue (0.9–1.2% fw).     

Inheritance and molecular mapping of a downy mildew resistance gene, Pl 13 in cultivated sunflower (Helianthus annuus L.)

The inheritance of resistance to sunflower downy mildew (SDM) derived from HA-R5 conferring resistance to nine races of the pathogen has been determined and the new source has been designated as Pl ₁₃ . The F₂ individuals and F₃ families of the cross HA-R5 (resistant) × HA 821 (susceptible) were screened against the four predominant SDM races 300, 700, 730, and 770 in separate tests which indicated dominant control by a single locus or a cluster of tightly linked genes. Bulked segregant analysis (BSA) was carried out on 116 F₂ individuals with 500 SSR primer pairs that resulted in the identification of 10 SSR markers of linkage groups 1 (9 markers) and 10 (1 marker) of the genetic map (Tang et al. in Theor Appl Genet 105:1124–1136, 2002) that distinguished the bulks. Of these, the SSR marker ORS 1008 of linkage group 10 was tightly linked (0.9 cM) to the Pl ₁₃ gene. Genotyping the F₂ population and linkage analysis with 20 polymorphic primer pairs located on linkage group 10 failed to show linkage of the markers with downy mildew resistance and the ORS 1008 marker. Nevertheless, validation of polymorphic SSR markers of linkage group 1 along with six RFLP-based STS markers of linkage group 12 of the RFLP map of Jan et al. (Theor Appl Genet 96:15–22, 1998) corresponding to linkage group 1 of the SSR map, mapped seven SSR markers (ORS 965-1, ORS 965-2, ORS 959, ORS 371, ORS 716, and ORS 605) including ORS 1008 and one STS marker (STS10D6) to linkage group 1 covering a genetic distance of 65.0 cM. The Pl ₁₃ gene, as a different source with its location on linkage group 1, was flanked by ORS 1008 on one side at a distance of 0.9 cM and ORS 965-1 on another side at a distance of 5.8 cM. These closely linked markers to the Pl ₁₃ gene provide a valuable basis for marker-assisted selection in sunflower breeding programs.     

Validity of <Emphasis Type="Italic">Scolecenchelys aoki</Emphasis>, with a redescription of <Emphasis Type="Italic">Scolecenchelys gymnota</Emphasis> (Anguilliformes: Ophichthidae)

The myrophine ophichthid fishes (worm eels) Muraenichthys aoki Jordan and Snyder 1901 and Muraenichthys gymnotus Bleeker 1857 are redescribed as valid species of Scolecenchelys based on the types and non-type specimens collected from the Indo-Pacific. Because both species are similar to each other in having acute snouts, the posterior margin of the eye before the rictus, and their dorsal-fin origins located slightly posterior to a vertical line through the anus, Scolecenchelys aoki has usually been regarded as a junior synonym of Scolecenchelys gymnota. However, S. aoki is clearly distinguishable from S. gymnota by having a median groove on the ventral side of snout (absent in S. gymnota), uniserial maxillary teeth in smaller specimens (<200 mm TL; vs. biserial), three infraorbital sensory pores at postorbital area (vs. two), and more numerous vertebrae (56–65 in predorsal vs. 51–57; 53–58 in preanal vs. 47–52). Scolecenchelys aoki is restricted to Japanese waters and regarded as a senior synonym of Muraenichthys borealis Machida and Shiogaki 1990. Scolecenchelys gymnota is widely distributed in the Indo-Pacific, from South Africa and the Red Sea to Samoa, north to Okinawa, Japan. Sphagebranchus huysmani Weber 1913 and Muraenichthys fowleri Schultz 1943 are synonymized under S. gymnota.     

Differentially expressed genes in embryonic cardiac tissues of mice lacking <Emphasis Type="Italic">Folr1</Emphasis>gene activity

Background  

Heart anomalies are the most frequently observed among all human congenital defects. As with the situation for neural tube defects (NTDs), it has been demonstrated that women who use multivitamins containing folic acid peri-conceptionally have a reduced risk for delivering offspring with conotruncal heart defects [1–3]. Cellular folate transport is mediated by a receptor or binding protein and by an anionic transporter protein system. Defective function of the Folr1 (also known as Folbp1; homologue of human FRα) gene in mice results in inadequate transport, accumulation, or metabolism of folate during cardiovascular morphogenesis.     

Rapid and repetitive plant regeneration of <Emphasis Type="Italic">Aralia elata</Emphasis> Seem. via somatic embryogenesis

In this work, we established a rapid and repetitive plant regeneration system for Aralia elata Seem. via primary and secondary somatic embryogenesis. Primary somatic embryogenesis was induced using leaf disks, petiole, and root segments, individually cultured for 5 weeks on Schenk and Hildebrandt (SH) (1972) medium with 0–5.0 mg/l indolebutyric acid (IBA). Our investigation demonstrated that optimal IBA concentrations of 3.0, 2.0, and 0.3 mg/l resulted in 100% somatic embryogenesis rates and averages of 11.3, 10.0, and 8.6 somatic embryos per explant for leaf disks, petiole, and root segments, respectively. The primary somatic embryos were used to conduct secondary somatic embryogenesis and the following treatments, in a gradient series, were examined: 0.3–4.0 mg/l IBA, 10–70 g/l sucrose and 0.2–3.0 mg/l abscisic acid (ABA). The results indicated that IBA was more effective than sucrose and ABA, and 3.0 mg/l IBA was the most suitable concentration for secondary somatic embryogenesis. Histological preparations indicated a multi-cellular origin of secondary somatic embryos and different morphological developmental stages during secondary somatic embryogenesis. Primary and secondary somatic embryos germinated readily and developed into normal plantlets after 2 weeks in woody plant medium (WPM, Lloyd and McCown 1980) with 20 g/l sucrose. At 4–5 cm in length, plantlets were transferred to soil (1:1 v/v of peat moss and sand) and the survival rate was 89% after 4 weeks under greenhouse conditions. This system provides a viable contribution to A. elata gene transformation, breeding and regeneration.     

Callus formation,plant regeneration,and transient expression in the halophyte sea aster (<Emphasis Type="Italic">Aster tripolium</Emphasis> L.)

An in vitro regeneration and transient expression systems were developed for the halophyte sea aster (Aster tripolium L.), an important genetic resource for salt tolerance. Adventitious shoots were formed from both leaf explants and suspension-cultured cells in a Murashige and Skoog (MS) (Physiol Plant 15:473–497, 1962) basal salts containing 500 mg l⁻¹ casamino acids, and supplemented with 5.4 μM a-naphthaleneacetic acid (NAA) and 4.7 μM kinetin to the culture medium. Hyperhydricity of shoots was avoided by increasing the ventilation of the culture vessel. Root formation from shoots was promoted in the presence of 26.9 μM NAA. A high yield of protoplasts was isolated using 1% cellulase and 0.25% pectinase from both leaf mesophyll and suspension-cultured cells, and these were used for transient expression. The highest level of transient expression of the green fluorescent protein was obtained with 1 × 10⁵ protoplasts ml⁻¹, 25 μg batch⁻¹ of plasmid vector, and 30% polyethylene glycol 4,000.     

Male Mating Interest Varies with Female Fecundity in <Emphasis Type="Italic">Pan troglodytes schweinfurthii</Emphasis> of Kanyawara,Kibale National Park

Female chimpanzees mate promiscuously during a period of extended receptivity marked by prominent sexual swelling. Recent studies of wild chimpanzees indicate that subtle variations in swelling size could act as a reliable cue of female fertilization potential both within and between cycles (Emery and Whitten Behavioral Ecology and Sociobiology, 54, 340–351, 2003; Deschner et al. Hormones and Behavior, 46, 204–215, 2004). Copulation rates increase during the periovulatory period and during conception cycles (Deschner et al. Hormones and Behavior, 46, 204–215, 2004; Emery Thompson American Journal of Primatology, 67, 137–158, 2005a), suggesting that males may be able to assess female fertilization potential. We asked whether facultative timing of copulation in Kanyawara chimpanzees was due to increased male mating interest or to increased female proceptivity during the most fecund days. We assessed multiple measures of male mating effort in cycles aligned relative to the day of detumescence and compared periovulatory days to other days of maximal swelling, and conception cycles to nonconception cycles. The rate and proportion of male initiative in soliciting sexual behavior increased during periods of highest fertilization potential. Males were also more likely to interrupt copulations, associate with estrous females, and compete with other males when females were most likely to conceive. Females initiated copulations more frequently during conception cycles but did not visibly shift mating behavior within cycles. Our results support the hypothesis that male chimpanzees have the ability to assess the profitability of mating attempts, a trait that may act as a counter-adaptation to female strategies to obscure paternity. We discuss potential cues and the implications for female reproductive strategies.     

Recombination suppression at the dominant <Emphasis Type="Italic">Rhg1/Rfs2</Emphasis> locus underlying soybean resistance to the cyst nematode

Host resistance to “yellow dwarf” or “moonlight” disease cause by any population (Hg type) of Heterodera glycines I., the soybean cyst nematode (SCN), requires a functional allele at rhg1. The host resistance encoded appears to mimic an apoptotic response in the giant cells formed at the nematode feeding site about 24–48 h after nematode feeding commences. Little is known about how the host response to infection is mediated but a linked set of 3 genes has been identified within the rhg1 locus. This study aimed to identify the role of the genes within the locus that includes a receptor-like kinase (RLK), a laccase and an ion antiporter. Used were near isogeneic lines (NILs) that contrasted at their rhg1 alleles, gene-based markers, and a new Hg type 0 and new recombination events. A syntenic gene cluster on Lg B1 was found. The effectiveness of SNP probes from the RLK for distinguishing homolog sequence variants on LgB1 from alleles at the rhg1 locus on LgG was shown. The resistant allele of the rhg1 locus was shown to be dominant in NILs. None of the recombination events were within the cluster of the three candidate genes. Finally, rhg1 was shown to reduce the plant root development. A model for rhg1 as a dominant multi-gene resistance locus based on the developmental control was inferred.     

Purification of the photosynthetic reaction center from <Emphasis Type="Italic">Heliobacterium modesticaldum</Emphasis>

We have developed a purification protocol for photoactive reaction centers (HbRC) from Heliobacterium modesticaldum. HbRCs were purified from solubilized membranes in two sequential chromatographic steps, resulting in the isolation of a fraction containing a single polypeptide, which was identified as PshA by LC–MS/MS of tryptic peptides. All polypeptides reported earlier as unknown proteins (in Heinnickel et al., Biochemistry 45:6756–6764, 2006; Romberger et al., Photosynth Res 104:293–303, 2010) are now identified by mass spectrometry to be the membrane-bound cytochrome c ₅₅₃ and four different ABC-type transporters. The purified PshA homodimer binds the following pigments: 20 bacteriochlorophyll (BChl) g, two BChl g′, two 8¹-OH-Chl a _F, and one 4,4′-diaponeurosporene. It lacks the PshB polypeptide binding the F_A and F_B [4Fe–4S] clusters. It is active in charge separation and exhibits a trapping time of 23 ps, as judged by time-resolved fluorescence studies. The charge recombination rate of the P₈₀₀ ⁺F_X⁻ state is 10–15 ms, as seen before. The purified HbRC core was able to reduce cyanobacterial flavodoxin in the light, exhibiting a K _M of 10 μM and a k _cat of 9.5 s⁻¹ under near-saturating light. There are ~1.6 menaquinones per HbRC in the purified complex. Illumination of frozen HbRC in the presence of dithionite can cause creation of a radical at g = 2.0046, but this is not a semiquinone. Furthermore, we show that high-purity HbRCs are very stable in anoxic conditions and even remain active in the presence of oxygen under low light.     

A Computational Analysis of Localized Ca<Superscript>2+</Superscript>-Dynamics Generated by Heterogeneous Release Sites

We investigate the role of heterogeneous expression of IP₃R and RyR in generating diverse elementary Ca²⁺ signals. It has been shown empirically (Wojcikiewicz and Luo in Mol. Pharmacol. 53(4):656–662, 1998; Newton et al. in J. Biol. Chem. 269(46):28613–28619, 1994; Smedt et al. in Biochem. J. 322(Pt. 2):575–583, 1997) that tissues express various proportions of IP₃ and RyR isoforms and this expression is dynamically regulated (Parrington et al. in Dev. Biol. 203(2):451–461, 1998; Fissore et al. in Biol. Reprod. 60(1):49–57, 1999; Tovey et al. in J. Cell Sci. 114(Pt. 22):3979–3989, 2001). Although many previous theoretical studies have investigated the dynamics of localized calcium release sites (Swillens et al. in Proc. Natl. Acad. Sci. U.S.A. 96(24):13750–13755, 1999; Shuai and Jung in Proc. Natl. Acad. Sci. U.S.A. 100(2):506–510, 2003a; Shuai and Jung in Phys. Rev. E, Stat. Nonlinear Soft Matter Phys. 67(3 Pt. 1):031905, 2003b; Thul and Falcke in Biophys. J. 86(5):2660–2673, 2004; DeRemigio and Smith in Cell Calcium 38(2):73–86, 2005; Nguyen et al. in Bull. Math. Biol. 67(3):393–432, 2005), so far all such studies focused on release sites consisting of identical channel types. We have extended an existing mathematical model (Nguyen et al. in Bull. Math. Biol. 67(3):393–432, 2005) to release sites with two (or more) receptor types, each with its distinct channel kinetics. Mathematically, the release site is represented by a transition probability matrix for a collection of nonidentical stochastically gating channels coupled through a shared Ca²⁺ domain. We demonstrate that under certain conditions a previously defined mean-field approximation of the coupling strength does not accurately reproduce the release site dynamics. We develop a novel approximation and establish that its performance in these instances is superior. We use this mathematical framework to study the effect of heterogeneity in the Ca²⁺-regulation of two colocalized channel types on the release site dynamics. We consider release sites consisting of channels with both Ca²⁺-activation and inactivation (“four-state channels”) and channels with Ca²⁺-activation only (“two-state channels”) and show that for the appropriate parameter values, synchronous channel openings within a release site with any proportion of two-state to four-state channels are possible, however, the larger the proportion of two-state channels, the more sensitive the dynamics are to the exact spatial positioning of the channels and the distance between channels. Specifically, the clustering of even a small number of two-state channels interferes with puff/spark termination and increases puff durations or leads to a tonic response.