A novel missense mutation of $$\textit{}$$ gene in a Chinese family leading to dyschromatosis symmetrica hereditaria and literature review

Dyschromatosis symmetrica hereditaria (DSH) is a rare autosomal dominant pigmentary genodermatosis, which is characterized by a mixture of hyperpigmented and hypopigmented macules on the dorsal of the hands and feet, and on the face presented like freckle. Identification of RNA-specific adenosine deaminase 1 (ADAR1) gene results in DSH. This study was mainly to explore the pathogenic mutation of ADAR1 gene and provide genetics counselling and prenatal diagnostic testing for childbearing individuals. Mutational analysis of ADAR1 gene was performed by polymerase chain reaction (PCR) and electrophoretic separation of PCR products by 1.5% agarose gel electrophoresis. The coding exons and intron/exon flanking regions followed by bidirectional sequencing was performed on all participants. In this study, we found that a 28 year-old male patient harbouring a deleterious substitution of Leu1052Pro in the ADAR1 gene in a typical DSH family. His mother suffered from the DSH also owns the same mutation. This mutation, however, is not identified in the unaffected members in this family and those 200 normal controls. In summary, this new mutation Leu1052Pro reported here is pathogenic and detrimental for DSH. Our finding not only enriches mutation database and contributes to dissecting further the correlation between mutation position and phenotypical features of DSH, but also provides genetics counselling and prenatal diagnostic testing for childbearing couple.     

Ultrastructural studies and molecular characterization of root-associated fungi of $$\textit{}$$ (D. Don) Szlach.: a threatened and medicinally important taxon

Crepidium acuminatum (Orchidaceae) is a threatened medicinal orchid that grows under shady and moist forest floor where light remains for a very short period of time. Mycorrhizal association is known to be essential for seed germination and seedling establishment in a majority of orchids. Identification of fungi that form mycorrhizae with orchids is of crucial importance for orchid conservation. We used both morphological as well as molecular approaches to study this plant–fungal interaction. Scanning electron microscopy showed that fungi grow and proliferate in the middle layers of the cortex. Also, spiral-root hairs were found along with root hairs, which is an unusual observation. Spiral-root hairs provide more surface area for fluid absorption and entrance of colonizers. Further, total root genomic DNA was isolated and fungal internal-transcribed spacer (ITS) regions were polymerase chain reaction (PCR)-amplified using specific primer combinations ITS1F/ITS4 and ITS1/ITS4tul. ITS sequences were obtained and analysed to know the closest sequence matche in the GenBank using BLASTn hosted by NLM-NCBI. Subject sequences were identified to be belonging to three main genera, namely, Tulasnella, Aspergillus and Penicillium. Results indicate that mycorrhizal association is necessary for the growth and development of the plant. In addition, this symbiosis influences the distribution and rarity of this medicinally valuable taxon. Specific fungal partners may lead to an enhanced seed germination rate and increased efficiency of nutrient exchange between both the partners. Hence, knowledge of mycorrhizal fungi is essential for future in vitro germination and seedling establishment programmes, because they rely on fungi for germination. Identification of mycorrhizal fungi can be used for orchid propagation and conservation programmes.     

Identification and mapping of molecular markers linked to the tuberculate fruit gene in the cucumber (Cucumis sativus L.)

Warty fruit is one of the highly valuable external quality traits related to the market values of cucumber. Genetic analysis has shown that a single dominant gene, Tu (Tuberculate fruit), determines the warty fruit trait in the cucumber plant. An F₂ population (247 individuals) from the cross of S06 × S52 was used for the mapping of the Tu/tu locus. By combining bulked segregant analysis with the sequence-related amplified polymorphism (SRAP) and simple sequence repeat (SSR) markers, 15 markers (9 SRAPs and 6 SSRs) linked to the Tu/tu locus were identified. Of nine SRAP markers, three closely linked to the Tu/tu locus were successfully converted into sequence characterized amplified region (SCAR) markers. The Tu/tu locus was mapped between the co-dominant SSR marker SSR16203 and the SCAR marker C_SC933, at a genetic distance of 1.4 and 5.9 cM, respectively. Then the linked SSR markers in the study were used as anchor loci to locate the Tu/tu locus on cucumber chromosome 5. Moreover, the validity analysis of the C_SC69 and C_SC24 markers was performed with 62 cucumber lines of diverse origins, showing that the two SCAR markers can be used for marker-assisted selection (MAS) of the warty fruit trait in cucumber breeding. The information provided in this study will facilitate the map-based cloning of the Tu/tu gene.     

Identification and characterization of gene-based SSR markers in date palm (Phoenix dactylifera L.)

Background

Clp/Hsp100 chaperones are involved in protein quality control. They act as independent units or in conjunction with a proteolytic core to degrade irreversibly damaged proteins. Clp chaperones from plant chloroplasts have been also implicated in the process of precursor import, along with Hsp70 chaperones. They are thought to pull the precursors in as the transit peptides enter the organelle. How Clp chaperones identify their substrates and engage in their processing is not known. This information may lie in the position, sequence or structure of the Clp recognition motifs.

Results

We tested the influence of the position of the transit peptide on the interaction with two chloroplastic Clp chaperones, ClpC2 and ClpD from Arabidopsis thaliana (AtClpC2 and AtClpD). The transit peptide of ferredoxin-NADP⁺ reductase was fused to either the N- or C-terminal end of glutathione S-transferase. Another fusion with the transit peptide interleaved between two folded proteins was used to probe if AtClpC2 and AtClpD could recognize tags located in the interior of a polypeptide. We also used a mutated transit peptide that is not targeted by Hsp70 chaperones (TP1234), yet it is imported at a normal rate. The fusions were immobilized on resins and the purified recombinant chaperones were added. After a washing protocol, the amount of bound chaperone was assessed. Both AtClpC2 and AtClpD interacted with the transit peptides when they were located at the N-terminal position of a protein, but not when they were allocated to the C-terminal end or at the interior of a polypeptide.

Conclusions

AtClpC2 and AtClpD have a positional preference for interacting with a transit peptide. In particular, the localization of the signal sequence at the N-terminal end of a protein seems mandatory for interaction to take place. Our results have implications for the understanding of protein quality control and precursor import in chloroplasts.     

水稻长穗颈基因eui紧密连锁SSR标记获得

02428h是从半矮秆材料02428体细胞培养后代中发现的隐性高秆突变体, 其株高性状由1对长穗颈基因eui和1对半矮秆基因sd-1共同控制。以02428h与半矮秆材料南京11杂交的F₂为作图群体, 利用Gramene公布的SSR标记和根据NCBI中的BAC序列自己新开发的SSR标记, 将eui基因定位在第5染色体上的RM3673和RM0012之间, 两侧遗传距离分别为0.3 cM和1.0 cM, 为该基因的分子标记辅助选择奠定了基础。     

Molecular mapping of a nuclear male-sterility gene in sunflower (Helianthus annuus L.) using TRAP and SSR markers

A nuclear male-sterile mutant, NMS 360, induced by streptomycin from an inbred maintainer line HA 89, possesses a single recessive gene, ms9, controlling male sterility. The present study identified DNA markers linked to the ms9 gene in an F₂ population derived from the cross of NMS 360 × RHA 271 and maps the ms9 gene to an existing sunflower SSR linkage map. Bulked segregant analysis was performed using the target region amplification polymorphism (TRAP) marker technique and the simple sequence repeats (SSR) technique. From 444 primer combinations, six TRAP markers linked with the ms9 gene were amplified. Two markers, Ts4p03-202 and Tt3p09-529, cosegregated with the ms9 gene. The other four markers, To3d14-310, Tt3p17-390, Ts4p23-300, and Tt3p09-531, linked with ms9 at a distance of 1.2, 3.7, 10.3, and 22.3 cM, respectively. Thirty SSR primers from 17 linkage groups of a PHA × PHB cultivated sunflower linkage map were screened among the two parents and the F₂ population. SSR primer ORS 705 of linkage group 10 was tightly linked to ms9 at a distance of 1.2 cM. The ms9 gene was subsequently mapped to linkage group 10 of the public sunflower SSR linkage map. The markers that were tightly linked with the ms9 gene will be useful in marker-assisted selection of male-sterile plants among segregating populations, and will facilitate the isolation of the ms9 gene by map-based cloning.     

Identification of DNA markers linked to the male sex in dioecious hemp (Cannabis sativa L.)

 A 400-bp RAPD marker generated by a primer of random decamer sequence has been found associated with the male sex phenotype in 14 dioecious cultivars and accessions of hemp (Cannabis sativa L.). The primer OPA8 generates a set of bands, most of which polymorphic among all the individual plants tested, and 1 of which, named OPA8₄₀₀, present in all male plants and absent in female plants. A screening of 167 plants belonging to different genotypes for the association of the OPA8₄₀₀ marker with the sex phenotype revealed that only in 3 cases was the 400-bp band was present in plants phenotypically female; on the contrary, in male plants the band was never missing, while in monoecious plants it was never present. Despite this sex-specific association, the sequences corresponding to OPA8₄₀₀ were present in both staminate and carpellate plants, as revealed by Southern blotting and hybridization with the cloned RAPD band. The RAPD marker was sequenced, and specific primers were constructed. These primers generated, on the same genotypes used for RAPD analysis, a SCAR marker 390 bp in length and male-specific. This SCAR is suitable for a precise, early and rapid identification of male plants during breeding programs of dioecious and monoecious hemp. Received: 16 January 1998 / Accepted: 30 April 1998     

Identification of molecular markers linked to trilocular gene (mc1) in Brassica juncea L.

Multilocular plants generally have higher yield than bilocular plants, which is attributable to the difference in the number of seeds per silique. In this study, we investigated a landrace of Brassica juncea in China that has trilocular siliques. We discovered that two independent recessive genes (mc1 and mc2) controlled the trait of trilocular silique. Mc1 was preliminarily mapped using amplified fragment length polymorphism (AFLP) technology in combination with bulked segregant analysis. From a survey of 2,048 AFLP primer combinations, we obtained 24 AFLP markers linked to the target gene, of which five were successfully converted into sequence-characterized amplified region markers. Two of these five AFLP markers and two other AFLP markers shared high sequence homology with A07 in the Brassica rapa genome. We developed seven simple sequence repeat primer pairs based on their sequence homology with A07. Our result also demonstrated that EC14MC14 and SC20 are the closest markers flanking the Mc1 gene, at distances of 1.1 and 1.6 cM, respectively. These molecular markers will facilitate the fine mapping and cloning of the mc1 gene and accelerate the selection process for multilocular rapeseed through marker-assisted selection.     

Identification and mapping of chi115 gene and DNA markers linked to it in pea (Pisum sativum L.)

Chlorophyll mutant Chi115 was induced by ethylmethane sulfonate (EMS) treatment of seeds of genotype Torsdag in Moscow State University and is characterized by lighter plant color. The monogenic nature of the mutant was determined by analyzing the F2 population from a cross between two P. sativum genotypes, WL1238 and Chi115. To establish a local map around the chi115 gene, the RAPD and ISSR techniques were used with 45 RAPD and 10 ISSR primers in combination with bulked segregant analysis (BSA). Linkage of 12 RAPDs and 2 ISSRs to the chi115 locus was observed in analysis of F2 single plants. Two RAPD markers that were closely associated with the chi115 gene were converted into the sequence characterized amplified region (SCAR) markers. By lowering the LOD score to 2, the linkage group containing the chi115 gene could be linked to the b gene (color of the flower) on linkage group III. Nevertheless, to prove the result obtained, three CAPS markers Sodmt, TubA1, and Rb were chosen on linkage group III. The results of linkage analysis showed that these CAPS markers were located within the linkage group including the chi115 gene.     

Assessment of genetic variation in tomato （Solanum lycopersicum L.） inbred lines using SSR molecular markers

A study was conducted to determine the genetic diversity of 39 determinate and indeterminate tomato inbred lines collected from China, Japan, S. Korea, and USA. Using 35 SSR polymorphic markers, a total of 150 alleles were found with moderate levels of diversity, and a high number of unique alleles existing in these tomato lines. The mean number of alleles per locus was 4.3 and the average polymorphism information content (PIC) was 0.31. Unweighted Pair Group Method with Arithmetic Mean (UPGMA) clustering at genetic similarity value of 0.85 grouped the inbred lines into four groups, where one USA cultivar formed a separate and more distant cluster. The most similar inbred lines are from USA, both with determinate type, whereas the most different lines are from USA (Us-16) and Japan (Ja-2) with determinate and indeterminate growth habit, respectively. Clustering was consistent with the known information regarding geographical location and growth habit. The genetic distance information reported in this study might be used by breeders when planning future crosses among these inbred lines.     

基于形态和分子特征的中华白蛉分类研究（双翅目：毛蠓科）

为准确鉴别中华白蛉,探讨其分类地位,对采自陕西、河南、四川和甘肃的中华白蛉,以及新疆的吴氏白蛉、长管白蛉进行了形态学观察和分子特征分析。检视了成虫的形态鉴别特征,测定和分析了其核糖体DNA第2内转录间隔区(rDNA-ITS2)和线粒体DNA细胞色素b(mtDNA-cyt b)基因部分序列。结果显示各地中华白蛉形态变异小,高海拔地区的个体仅翅较长。ITS2和Cyt b序列在中华白蛉种内均具异质性,但其间的差异程度能较好地解析白蛉属部分种间的关系。本研究结果提示中华白蛉种群在形态和分子水平已出现明显遗传分化。     

Identification of AFLP markers linked to resistance of cowpea (Vigna unguiculata L.) to parasitism by Striga gesnerioides

AFLP and bulked segregant analysis were used to identify molecular markers linked to resistance of cowpea [Vigna ungiculata (L.) Walp.] to parasitism by Striga gesnerioides (Willd.) Vatke. Segregation analysis of F₂ progeny from a cross of Tvx3236, a Striga-susceptible line, with IT82D-849, a resistant cultivar, showed that resistance to S. gesnerioides race 1 from Burkina Faso was controlled by a single dominant gene, designated Rsg2–1. Three AFLP markers were identified that are tightly linked to Rsg2–1: E-AAC/M-CAA₃₀₀ (2.6 cM), E-ACT/M-CAA₅₂₄ (0.9 cM), and E-ACA/M-CAT_140/150 (0.9 cM), which appears to be codominant. Segregation analysis of a different F₂ population resulting from a cross of the Striga-susceptible line IT84S-2246–4 with Tvu 14676, a S. gesnerioides race 3 resistant line, showed that resistance to S. gesnerioides race 3 was also controlled by a single dominant gene, designated Rsg4–3. Six AFLP markers linked to Rsg4–3 were identified: E-ACA/M-CAG₁₂₀ (10.1 cM), E-AGC/M-CAT₈₀ (4.1 cM), E-ACA/M-CAT₁₅₀ (2.7 cM), E-AGC/M-CAT₁₅₀ (3.6 cM), E-AAC/M-CAA₃₀₀ (3.6 cM), and E-AGC/M-CAT₇₀ (5.1 cM). Segregation analysis of the E-AAC/M-CAA₃₀₀ and E-ACA/M-CAG₁₂₀ markers in recombinant inbred lines derived from IT84S-2049×524B determined that both are located within linkage group 1 of the cowpea genetic map. The identification of AFLP markers linked to Striga resistance provides a stepping stone for a marker-assisted selection program and the eventual cloning and characterization of the gene(s) encoding resistance to this noxious parasitic weed. Received: 24 April 2000 / Accepted: 21 August 2000     

Development and use of novel SSR markers for molecular genetic diversity in Italian millet (Setaria italica L.)

Italian millet is a commercially important grain crop. Nineteen polymorphic simple sequence repeat (SSR) markers, developed through construction of an SSR-enriched library from genomic DNA of Italian millet (Setaria italica L., P. Beauv.), were used for assessment of molecular genetic diversity against 40 accessions of S. italica. In total, 85 alleles were detected, with an average of 4.5 alleles per locus. The average gene diversity and polymorphism information content (PIC) values were 0.412 and 0.376, ranging from 0.02 to 0.88 and from 0.02 to 0.87, respectively. Values for observed (H _O) and expected (H _E) heterozygosities ranged from 0 to 0.73 and from 0.03 to 0.89, respectively. Nine loci deviated from Hardy-Weinberg equilibrium. The mean similarity coefficient among accessions was 0.6593. Based on the UPGMA algorithm, six different groups were successfully identified. In this clustering analysis, all Korean accessions grouped in one cluster, indicating that Korean accessions are genetically quite distinct from other introduced accessions. These newly developed microsatellite markers should be very useful tools for several genetic studies, including an assessment of diversity and population structure in Italian millet.     

Assessment of variability among morphological and molecular characters in wild populations of mint [Mentha longifolia (L.) L.] germplasm

Mentha longifolia is an important medicinal and aromatic perennial herb that exhibits wide distribution range from sub-tropical to temperate regions. In the present study, agro-morphological traits and genetic differences in 19 different populations of M. longifolia were studied to evaluate the level and extent of its diversity. Analysis of variance (ANOVA) showed that the different phenotypic characters show considerable differences among various populations and was significant at p < 0.05. Molecular diversity analysis performed by using arbitrary amplified eleven ISSR primers generated a total of 121 amplicons that range within the size of 200–2500 base pairs (bp). Each primer on average generated 11 amplicons with percentage polymorphism being 100. The analysis of molecular variance (AMOVA) showed more (64%) among population genetic diversity and less (36%) within the populations. Greater genetic differentiation (Gst = 0.6852) among these populations occurs due to low gene flow (Nm = 0.2297) and greater habitat variability. Geographic and genetic distances were positively correlated according to Mantel’s test. In order to remove any kind of biases, we used R software to perform cluster and redundancy analysis to analyse the extent of relatedness among studied populations. In terms of morphological and molecular aspects, the populations were grouped into four and five clusters respectively based on hierarchical clustering method. The results demonstrated that M. longifolia displays a great degree of morphological and genetic variation and can be utilized in breeding, genetic improvement, and gene bank conservation programmes in future.     

Validation of SSR markers linked to null kunitz tryspin inhibitor allele in Indian soybean [Glycine max (L.) Merr.] population

Kunitz trypsin inhibitor, a proteinaceous antinutritional factor present in soybean seeds, is responsible for inferior nutritional quality of raw soybean and incompletely processed soy products. The objective of the present investigation was to validate the SSR markers (Satt228 and Satt409) reported to be linked to Ti locus in an Indian soybean population generated from the cross between soybean cultivar LSb1 (TiTi) and PI542044 (titi). Parental polymorphism was surveyed using Satt409, Satt228 and 5 SSR markers in the neighbouring genomic region of Ti locus. A portion of the cotyledon of F₂ seeds was used for analyzing the presence or absence of kunitz trypsin inhibitor polypeptide electrophoretically while the remaining portion containing the embryo was used for raising the F₂ plants (104) for the development of mapping population. The SSR marker Satt228 reported to be tightly linked with Ti locus was not found to be polymorphic for the parents used in our study. Satt409 was found to be linked with Ti locus at 4.7 cM. Besides, a new marker Satt538 was found to be linked with Ti locus at a distance of 17.8 cM. Thus, the SSR marker Satt409 can be useful for Marker Assisted Selection for transferring titi allele in the background of Indian soybean genotypes.     

Genetic diversity among varieties and wild species accessions of pea (Pisum sativum L.) based on molecular markers, and morphological and physiological characters.

Random amplified polymorphic DNA, simple sequence repeat, and inter-simple sequence repeat markers were used to estimate the genetic relations among 65 pea varieties (Pisum sativum L.) and 21 accessions from wild Pisum subspecies (subsp.) abyssinicum, asiaticum, elatius, transcaucasicum, and var. arvense. Fifty-one of these varieties are currently available for growers in western Canada. Nei and Li's genetic similarity (GS) estimates calculated using the marker data showed that pair-wise comparison values among the 65 varieties ranged from 0.34 to 1.00. GS analysis on varieties grouped according to their originating breeding programs demonstrated that different levels of diversity were maintained at different breeding programs. Unweighted pair-group method arithmetic average cluster analysis and principal coordinate analysis on the marker-based GS grouped the cultivated varieties separately from the wild accessions. The majority of the food and feed varieties were grouped separately from the silage and specialty varieties, regardless of the originating breeding programs. The analysis also revealed some genetically distinct varieties such as Croma, CDC Handel, 1096M-8, and CDC Acer. The relations among the cultivated varieties, as revealed by molecular-marker-based GS, were not significantly correlated with those based on the agronomic characters, suggesting that the 2 systems give different estimates of genetic relations among the varieties. However, on a smaller scale, a consistent subcluster of genotypes was identified on the basis of agronomic characters and their marker-based GS. Furthermore, a number of variety-specific markers were identified in the current study, which could be useful for variety identification. Breeding strategies to maintain or enhance the genetic diversity of future varieties are proposed.     

Identification of AFLP and microsatellite markers linked with an aluminium tolerance gene in barley ( Hordeum vulgare L.)

Barley is the most sensitive among the cereals to aluminium (Al) stress and breeding for more tolerant cultivars is a priority. To enhance selection efficiency for Al tolerance in barley, PCR-based AFLP and microsatellite markers linked to a locus conferring tolerance to aluminium were identified. The study used F(2) progeny derived from a single cross between Yambla (moderately tolerant of Al) and WB229 (tolerant of Al) and developed hydroponic pulse-recovery screening methods to assess tolerance of phenotypes based on root growth. The segregation ratios of tolerant and sensitive genotypes and F(3) progeny testing suggest that a single major gene controlled Al tolerance ( Alt). In order to determine the chromosomal location of the Alt gene, we used the AFLP technique coupled with bulk segregant analysis. We evaluated tolerant and sensitive bulks using 30 combinations of EcoRI/ MseI primers, and 12 of these permitted differentiation of the sensitive and tolerant bulks. More than 1,000 amplified fragments were obtained, and 98 polymorphic bands were scored. AFLP analysis of wheat-barley chromosome addition lines indicated that the Alt gene was located on barley chromosome 4H. Four chromosome 4H-specific microsatellite markers (Bmac310, Bmag353, HVM68 and HVMCABG) were tightly linked to Alt. The large allelic variation detected with microsatellite marker Bmag353 allowed us to implement this marker for routine marker-assisted selection for Al tolerance, and 396 plants could be screened on a single gel.     

Genetic linkage map of peach [Prunus persica (L.) Batsch] using morphological and molecular markers

A genetic linkage map of peach [Prunus persica (L.) Batch] was constructed in order to identify molecular markers linked to economically important agronomic traits that would be particularly useful for long-lived perennial species. An intraspecific F₂ population was generated from self-pollinating a single F₁ plant from a cross between a flat non-acid peach, ‘Ferjalou Jalousia^®’ and an acid round nectarine ‘Fantasia’. Mendelian segregations were observed for 270 markers including four agronomic characters (peach/nectarine, flat/round fruit, acid/non-acid fruit, and pollen sterility) and 1 isoenzyme, 50 RFLP, 92 RAPD, 8 inter-microsatellite amplification (IMA), and 115 amplified fragment length polymorphism (AFLP) markers. Two hundred and forty-nine markers were mapped to 11 linkage groups covering 712 centiMorgans (cM). The average density between pairs of markers is 4.5?cM. For the four agronomic characters studied, molecular markers were identified. This map will be used for the detection of QTL controlling fruit quality in peach and, particularly, the acid and sugar content.     

Association analysis,genetic diversity and population structure of barley (Hordeum vulgare L.) under heat stress conditions using SSR and ISSR markers linked to primary and secondary metabolites

Molecular Biology Reports - Barley is one of the major cereal crops, which can provide a significant source of genes for stress tolerance due to its high diversity and adaptability. Metabolite...