STS Markers Research Articles

Identification of bacterial blight resistance gene Xa7 in rice (Oryzae sativa L.) through STS marker

Bacterial blight caused by Xanthomonas Oryzae pv. Oryzae (Xoo) is the most destructive disease of rice that limits rice yield in all major rice-growing regions of Pakistan, especially in irrigated lowland conditions. Since bacterial pathogen is difficult to manage, development of host plant resistance is the most effective mean to control this disease. In this investigation, a major gene (Xa-7) conferring broad spectrum resistance to various races of the pathogen has been identified in various varieties and advance lines of rice by STMS marker. Out of 74 rice varieties, 31 to 44 showed the presence of Xa-7. Identification of Xa7 gene in rice could be utilized for increasing the level of resistance of existing rice varieties.

Identification of molecular markers linked to rice bacterial blight resistance genes from Oryza meyeriana

Y73 is a progeny of asymmetric somatic hybridization between Oryza sativa cv. Dalixiang and the wild rice species Oryza meyeriana. Inoculation with a range of strains of Xanthomonas oryzae pv. oryzae showed that Y73 had inherited a high level of resistance to rice bacterial blight (BB) from its wild parent. An F2 population of 7125 individuals was constructed from the cross between Y73 and a BB-susceptible cultivar IR24. After testing 615 SSR and STS markers covering the 12 rice chromosomes, 186 markers were selected that showed polymorphism between Y73 and IR24. Molecular markers linked to the BB resistance genes in Y73 were scanned using the F2 population and the polymorphic markers. The SSR marker RM128 on chromosome 1, the STS marker R03D159 on chromosome 3 and the STS marker R05D104 on chromosome 5 were found to be linked to the rice BB resistance genes in Y73.

Molecular mapping of a stripe rust resistance gene in wheat line C51.

Stripe rust, a major disease in areas where cool temperatures prevail, can strongly influence grain yield. To control this disease, breeders have incorporated seedling resistance genes from a variety of sources outside the primary wheat gene pool. The wheat line C51, introduced from the International Center for Agricultural Research in the Dry Areas (ICARDA), Syria, confers resistance to all races of Puccinia striiformis f. sp. tritici (PST) in China. To map the resistant gene(s) against stripe rust in wheat line C51, 212 F8 recombinant inbred lines (RILs) derived from the cross X440 x C51 were inoculated with Chinese PST race CYR33 (Chinese yellow rust, CYR) in the greenhouse. The result showed that C51 carried a single dominant gene for resistance (designated YrC51) to CYR33. Simple sequence repeat (SSR) and resistance gene-analogue polymorphism (RGAP) markers that were polymorphic between the parents were used for genotyping the 212 F8 RILs. YrC51 was closely linked to two SSR loci on chromosome 2BS with genetic distances of 5.1 cM (Xgwm429) and 7.2 cM (Xwmc770), and to three RGAP markers C51R1 (XLRR For / NLRR For), C51R2 (CLRR Rev / Cre3LR-F) and C51R3 (Pto kin4 / NLRRINV2) with genetic distances of 5.6, 1.6 and 9.2 cM, respectively. These RGAP-linked markers were then converted into STS markers.Among them, one STS marker, C51STS-4, was located at a genetic distance of 1.4 cM to YrC51 and was closely associated with resistance when validated in several populations derived from crosses between C51 and Sichuan cultivars. The results indicated that C51STS-4 can be used for marker assisted selection (MAS) and would facilitate the pyramiding of YrC51 with other genes for stripe rust resistance.

SEQUENCING OF THE PHYLLOXERA RESISTANCE LOCUS RDV1 OF CULTIVAR 'BÖRNER'

Cultivar ‘Borner’, an interspecific cross of Vitis riparia Gm 183 and V. cinerea Arnold, is a phylloxera root resistant rootstock showing neither tuberosity nor nodosity symptoms. The Rdv1 locus, a major quantitative trait locus (QTL) localized on chromosome 13, correlates with the resistance phenotype. In order to identify the responsible gene(s) for this trait a high density genetic map was established for this region deducing STS markers from the V. vinifera genome sequence. For physical mapping a bacterial artificial chromosome (BAC) library was screened with the STS markers of the Rdv1 locus. The isolated clones were used to build up a minimal tiling path for the V. cinerea haplotype covering almost the entire locus. The BACs were sequenced, contigs assembled and finishing accomplished using the PN40024 genome sequence as a reference. Small final gaps were closed by sequencing of PCR products. Comparison of the 350 kb Rdv1 sequence with the V. vinifera sequence of PN40024 revealed a conserved gene order even for the small RGA cluster located in the middle of the sequence of both genomes. However, between these RGA clusters a low sequence homology and larger structural rearrangements within the RGAs and repetitive transposon fragments were observed. Therefore, the NBS-LRR-like genes located in the RGA cluster of the Rdv1 locus are favourable candidate genes for the phylloxera resistance of rootstock ‘Borner’.

Fine physical and genetic mapping of powdery mildew resistance gene MlIW172 originating from wild emmer (Triticum dicoccoides).

Powdery mildew, caused by Blumeria graminis f. sp. tritici, is one of the most important wheat diseases in the world. In this study, a single dominant powdery mildew resistance gene MlIW172 was identified in the IW172 wild emmer accession and mapped to the distal region of chromosome arm 7AL (bin7AL-16-0.86-0.90) via molecular marker analysis. MlIW172 was closely linked with the RFLP probe Xpsr680-derived STS marker Xmag2185 and the EST markers BE405531 and BE637476. This suggested that MlIW172 might be allelic to the Pm1 locus or a new locus closely linked to Pm1. By screening genomic BAC library of durum wheat cv. Langdon and 7AL-specific BAC library of hexaploid wheat cv. Chinese Spring, and after analyzing genome scaffolds of Triticum urartu containing the marker sequences, additional markers were developed to construct a fine genetic linkage map on the MlIW172 locus region and to delineate the resistance gene within a 0.48 cM interval. Comparative genetics analyses using ESTs and RFLP probe sequences flanking the MlIW172 region against other grass species revealed a general co-linearity in this region with the orthologous genomic regions of rice chromosome 6, Brachypodium chromosome 1, and sorghum chromosome 10. However, orthologous resistance gene-like RGA sequences were only present in wheat and Brachypodium. The BAC contigs and sequence scaffolds that we have developed provide a framework for the physical mapping and map-based cloning of MlIW172.

Development and application of PCR markers specific to the 1Ns chromosome of Psathyrostachys huashanica Keng with leaf rust resistance

Wheat–Psathyrostachys huashanica Keng disomic addition line 12-3 was developed and characterized using genomic in situ hybridization (GISH), expressed sequence tag–sequence tagged site (EST–STS), and sequence characterized amplified region (SCAR) markers. Mitotic and meiotic GISH analyses indicated that it contained 42 wheat chromosomes and a pair of P. huashanica chromosomes. Eight EST–STS multiple-loci markers located on the homoeologous group 1 chromosomes of wheat amplified polymorphic bands in the 1Ns disomic addition line 12-3, which were unique to P. huashanica. These markers suggested that the introduced Ns chromosomes belonged to homoeologous group 1. Furthermore, diagnostic fragments of random amplified polymorphic DNA marker OPAG10986 and simple sequence repeat marker Xgwm601 135 were cloned, sequenced, and converted into SCAR markers, i.e., RHS153 and SHS10, respectively, which were validated using a range of distinct plant species and a complete set of wheat–P. huashanica disomic addition lines (1Ns–7Ns, 2n = 44 = 22 II). The results demonstrated that the SCAR markers targeted the Ns genome of P. huashanica and they were linked to the 1Ns chromosome. In addition, 12-3 was evaluated to test its leaf rust resistance in the adult stages and its agronomic traits. These newly developed EST–STS and SCAR markers will be powerful tools for wheat breeders who want to screen for genotypes containing the 1Ns chromosome, with low costs and high throughput.

TaGS-D1, an ortholog of rice OsGS3, is associated with grain weight and grain length in common wheat

The OsGS3 gene plays a principal role in controlling grain weight and grain length in rice. However, the function of an orthologous gene TaGS in wheat has not been analyzed to date. In the present study, we cloned the gDNA of TaGS gene, designated TaGS-D1, with four exons and three introns on chromosome 7DS by a comparative genomics approach. The cDNA of TaGS-D1 is 255 bp, and it encodes 85 amino acids. We also found a plant-specific organ size regulation domain in the deduced polypeptide, indicating that TaGS-D1, like OsGS3, does not belong to the PEBP family. DNA sequencing of the TaGS-D1 locus revealed no diversity in the coding sequence of exons, but there was a single nucleotide polymorphism (SNP) in the first intron, and 30 SNPs, a 40-bp InDel and a 3-bp InDel were found in the second intron between genotypes with higher and lower thousand grain weights (TGW). Based on the 40-bp InDel, a co-dominant STS marker, designated GS7D, was developed to discriminate the two alleles. GS7D was 8.0 cM from Xbarc184 located on chromosome 7DS by linkage mapping. A QTL for TGW and grain length at GS7D locus explained up to 16.3 and 7.7 %, respectively, of the phenotypic variances in a RIL population derived from Doumai/Shi 4185 grown in Shijiazhuang and Beijing. One hundred and seventy-five Chinese wheat cultivars were genotyped with GS7D, indicating that TaGS-D1 was significantly associated with grain weight. The allelic distribution at the TaGS-D1 locus showed that the frequencies of TaGS-D1a were high in cultivars from Serbia, Japan, Australia, Canada, and the Northeastern Spring Wheat and Northern Winter Wheat Regions of China.

Molecular mapping of an adult plant stem rust resistance gene Sr56 in winter wheat cultivar Arina.

This article covers detailed characterization and naming of QSr.sun - 5BL as Sr56 . Molecular markers linked with adult plant stem rust resistance gene Sr56 were identified and validated for marker-assisted selection. The identification of new sources of adult plant resistance (APR) and effective combinations of major and minor genes is well appreciated in breeding for durable rust resistance in wheat. A QTL, QSr.sun-5BL, contributed by winter wheat cultivar Arina providing 12-15% reduction in stem rust severity, was reported in an Arina/Forno recombinant inbred line (RIL) population. Following the demonstration of monogenic segregation for APR in the Arina/Yitpi RIL population, the resistance locus was formally named Sr56. Saturation mapping of the Sr56 region using STS (from EST and DArT clones), SNP (9K) and SSR markers from wheat chromosome survey sequences that were ordered based on synteny with Brachypodium distachyon genes in chromosome 1 resulted in the flanking of Sr56 by sun209 (SSR) and sun320 (STS) at 2.6 and 1.2cM on the proximal and distal ends, respectively. Investigation of conservation of gene order between the Sr56 region in wheat and B. distachyon showed that the syntenic region defined by SSR marker interval sun209-sun215 corresponded to approximately 192kb in B. distachyon, which contains five predicted genes. Conservation of gene order for the Sr56 region between wheat and Brachypodium, except for two inversions, provides a starting point for future map-based cloning of Sr56. The Arina/Forno RILs carrying both Sr56 and Sr57 exhibited low disease severity compared to those RILs carrying these genes singly. Markers linked with Sr56 would be useful for marker-assisted pyramiding of this gene with other major and APR genes for which closely linked markers are available.

High-resolution genetic mapping of rice bacterial blight resistance gene Xa23

Bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is the most devastating bacterial disease of rice (Oryza sativa L.), a staple food crop that feeds half of the world's population. In management of this disease, the most economical and effective approach is cultivating resistant varieties. Due to rapid change of pathogenicity in the pathogen, it is necessary to identify and characterize more host resistance genes for breeding new resistant varieties. We have previously identified the BB resistance (R) gene Xa23 that confers the broadest resistance to Xoo strains isolated from different rice-growing regions and preliminarily mapped the gene within a 1.7cm region on the long arm of rice chromosome 11. Here, we report fine genetic mapping and in silico analysis of putative candidate genes of Xa23. Based on F2 mapping populations derived from crosses between Xa23-containing rice line CBB23 and susceptible varieties JG30 or IR24, six new STS markers Lj36, Lj46, Lj138, Lj74, A83B4, and Lj13 were developed. Linkage analysis revealed that the new markers were co-segregated with or closely linked to the Xa23 locus. Consequently, the Xa23 gene was mapped within a 0.4cm region between markers Lj138 and A83B4, in which the co-segregating marker Lj74 was identified. The corresponding physical distance between Lj138 and A83B4 on Nipponbare genome is 49.8kb. Six Xa23 candidate genes have been annotated, including four candidate genes encoding hypothetical proteins and the other two encoding a putative ADP-ribosylation factor protein and a putative PPR protein. These results will facilitate marker-assisted selection of Xa23 in rice breeding and molecular cloning of this valuable R gene.

Developmental Changes of Recessive Genes-mediated Cucumber mosaic virus (CMV) Resistance in Peppers (Capsicum annuum L.)

Cucumber mosaic virus (CMV) is one of the most important viral diseases in pepper (Capsicum annuum L.), and several genes for resistance were reported in Capsicum spp. In Korea, a single dominant gene that is resistant to <TEX>$CMV_{Fny}$</TEX> and <TEX>$CMV_{P0}$</TEX> has been used for breeding. Recently, a new strain (<TEX>$CMV_{P1}$</TEX>) was reported that could infect cultivars resistant to both <TEX>$CMV_{Fny}$</TEX> and <TEX>$CMV_{P0}$</TEX>. Therefore, breeding of more robust CMV-resistant cultivars is required. In this study, we surveyed the inheritance of <TEX>$CMV_{P1}$</TEX> resistance and analyzed the location of the resistance loci. After <TEX>$CMV_{P1}$</TEX> inoculation of various germplasms and breeding lines, one accession (ICPN18-8) showed no visual symptoms at 15 dpi (days post inoculation) but was susceptible after 45 dpi, and one resistant line (I7339) showed resistance until at 45 dpi. The latter line was used for tests of resistance inheritance. A total of 189 <TEX>$F_2$</TEX> plants were examined, with 42 individuals showing resistance at 15 dpi and a phenotype segregation ratio close to 1:3 (resistant:susceptible plants). In a lateral ELISA test at 45 dpi, 11 plants showed resistance, and the segregation ratio was changed to 1:15. These results indicate that resistance in C. annuum 'I7339' is controlled by two different recessive genes; we named these resistance genes 'cmr3E' and 'cmr3L,' respectively. To locate these two resistant loci in the pepper linkage map, various RAPD, SSR, and STS markers were screened; only nine markers were grouped into one linkage group (LG). Only one RAPD primer (OPAT16) was distantly linked with cmr3E (22.3 cM) and cmr3L (20.7 cM). To develop more accurate markers for marker-assisted breeding, enriching for molecular markers spanning two loci will be required.

Genetic Rearrangements of Six Wheat–Agropyron cristatum 6P Addition Lines Revealed by Molecular Markers

Agropyron cristatum (L.) Gaertn. (2n = 4x = 28, PPPP) not only is cultivated as pasture fodder but also could provide many desirable genes for wheat improvement. It is critical to obtain common wheat–A. cristatum alien disomic addition lines to locate the desired genes on the P genome chromosomes. Comparative analysis of the homoeologous relationships between the P genome chromosome and wheat genome chromosomes is a key step in transferring different desirable genes into common wheat and producing the desired alien translocation line while compensating for the loss of wheat chromatin. In this study, six common wheat–A. cristatum disomic addition lines were produced and analyzed by phenotypic examination, genomic in situ hybridization (GISH), SSR markers from the ABD genomes and STS markers from the P genome. Comparative maps, six in total, were generated and demonstrated that all six addition lines belonged to homoeologous group 6. However, chromosome 6P had undergone obvious rearrangements in different addition lines compared with the wheat chromosome, indicating that to obtain a genetic compensating alien translocation line, one should recombine alien chromosomal regions with homoeologous wheat chromosomes. Indeed, these addition lines were classified into four types based on the comparative mapping: 6PI, 6PII, 6PIII, and 6PIV. The different types of chromosome 6P possessed different desirable genes. For example, the 6PI type, containing three addition lines, carried genes conferring high numbers of kernels per spike and resistance to powdery mildew, important traits for wheat improvement. These results may prove valuable for promoting the development of conventional chromosome engineering techniques toward molecular chromosome engineering.

Mapping of STS markers developed from drought tolerance candidate genes and preliminary analysis of their association with yield-related traits in common wheat(Triticum aestivum)

Drought is a severe abiotic stress that affects wheat production worldwide. In order to identify candidate genes for tolerance to water stress in wheat, sequences of 11 genes that have function of drought tolerance in other plant species were used to identify the wheat ortholog genes via homology searching in the wheat EST database. Atotal of 11 primer pairs were identified and amplified PCR products in wheat. Of them, 10 STS markers were mapped on 11 chromosomes in a set of nulli-tetrasomic lines of ‘Chinese Spring’ wheat; six were mapped on chromosomes 1A, 1B, 4B, 7A, 2B and 5D, respectively, in a spring wheat mapping population (POP1). The marker XTaABH1 mapped on 7A in POP1 was the only one mapped but characterized in a winter wheat mapping population (POP2) for grain yield, kernel weight and diameter, and height in four-field trials applied different water stress or irrigation. The marker XTaABH1 was significantly associated with grain yield under rainfed condition, with kernel weight under terminal stress and non-irrigation conditions, with kernel diameter and height under non-irrigated condition. The STS primers, map information and marker-trait association produced in the currently study would be of interest to researchers working on drought tolerance.

Development of STS and CAPS markers for variety identification and genetic diversity analysis of tea germplasm in Taiwan.

BackgroundTea (Camellia sinensis) is an important economic crop in Taiwan. Particularly, two major commercial types of tea (Paochong tea and Oolong tea) which are produced in Taiwan are famous around the world, and they must be manufactured with specific cultivars. Nevertheless, many elite cultivars have been illegally introduced to foreign countries. Because of the lower cost, large amount of “Taiwan-type tea” are produced and imported to Taiwan, causing a dramatic damage in the tea industry. It is very urgent to develop the stable, fast and reliable DNA markers for fingerprinting tea cultivars in Taiwan and protecting intellectual property rights for breeders. Furthermore, genetic diversity and phylogenetic relationship evaluations of tea germplasm in Taiwan are imperative for parental selection in the cross-breeding program and avoidance of genetic vulnerability.ResultsTwo STS and 37 CAPS markers derived from cytoplasmic genome and ESTs of tea have been developed in this study providing a useful tool for distinguishing all investigated germplasm. For identifying 12 prevailing tea cultivars in Taiwan, five core markers, including each one of mitochondria and chloroplast, and three nuclear markers, were developed. Based on principal coordinate analysis and cluster analysis, 55 tea germplasm in Taiwan were divided into three groups: sinensis type (C. sinensis var. sinensis), assamica type (C. sinensis var. assamica) and Taiwan wild species (C. formosensis). The result of genetic diversity analysis revealed that both sinensis (0.44) and assamica (0.41) types had higher genetic diversity than wild species (0.25). The close genetic distance between the first (Chin-Shin-Oolong) and the third (Shy-Jih-Chuen) prevailing cultivars was found, and many recently released varieties are the descents of Chin-Shin-Oolong. This implies the potential risk of genetic vulnerability for tea cultivation in Taiwan.ConclusionsWe have successfully developed a tool for tea germplasm discrimination and genetic diversity analysis, as well as a set of core markers for effective identification of prevailing cultivars in Taiwan. According to the results of phylogenetic analysis on prevailing tea cultivars, it is necessary to broaden genetic diversity from wild species or plant introduction in future breeding programs.Electronic supplementary materialThe online version of this article (doi:10.1186/1999-3110-55-12) contains supplementary material, which is available to authorized users.

Identification of Wheat Stripe Rust Resistance Genes in Iranian Wheat Cultivars Using Molecular Markers

Yellow or stripe rust is one of the most important and destructive wheat diseases all over the world. The best strategy to control this disease is genetic resistance through combining several resistance genes which results in achieving long lasti ng resistance. Marker assisted selection has provided a suitable means towards this strategy. The aim of this study was to identify the race specific seedling genes Yr5 and Yr10 and the race non- specific APR geneYr29 in a selection of 40 Iranian genotypes using STS and SSR markers. Therefore, genomic DNA was extracted from these genotypes, the susceptible cultivar AvocetSas negative control, and the genotypes with corresponding resistance gene (positive controls). PCR was performed using YrSTS7/8, Xpsp 3000 and Xwmc44 markers forYr5,Yr10 and Yr29, respectively. The results indicated the presence of Yr5in only 6 genotypes. The presence of a 260 bps band also showed that Yr10 was present in 12 genotypes whileYr29 was present in 13 cultivars. As all the

Is Diabetic Male Fertility Associated With Y- Chromosome Microdeletion? A Pilot Study

Decline in fertility rate is alarming and it affects about 15.0 % of couples trying to conceive. Globally, 5.0-7.0% of the general male population is affected by infertility and the trend may increase in future. Diabetes mellitus, a state of chronic hyperglycemia, is found to be an effective cause associated with male infertility. Since both type I and type II diabetes influences male infertility, we aimed to identify the genetic link underlying diabetic male infertility. Y chromosome microdeletions were screened in 12 Type I & Type II diabetic infertile male patients of varying seminogram profile by employing Triplex (sY84, sY99 and sY156) and individual (sY86, sY100, sY134 and sY254) PCR. sY156 STS marker of AZFc region was invariably deleted in 5 diabetic infertile men in addition to sY86, sY100, sY134 STS markers. No functional gene has been assigned for the 923bp DNA fragment encoded by the sY156 STS marker. Rather, it belongs to a processed pseudogene TRIM60P8Y. Frequent deletion of sY156 marker in AZFc region in diabetic infertile men is unclear. The result showed can be further substantiated with more number of samples.

Development and validation of molecular markers linked with stem rust resistance gene Sr13 in durum wheat

Stem rust resistance gene Sr13, found frequently in tetraploid wheats, was tested effective against Puccinia graminis f. sp. tritici pathotype Ug99 (TTKSK) and its derivatives. It remains a candidate for developing new cultivars with diverse combinations of stem rust resistance genes. To combine Sr13 with other genes that produce a similar phenotype, linked markers would be required. We used the AFLP approach to identify markers linked closely with Sr13. The STS marker AFSr13, derived from an AFLP fragment, mapped at 3.4–6.0 cM proximal to Sr13 across three mapping populations. Marker dupw167, previously reported to be linked with Sr13, mapped 2.3–5.7 cM distal to Sr13 in four F3 populations. Marker gwm427 mapped proximal to AFSr13 in two populations, and these markers were monomorphic on one population each. The map order dupw167–Sr13–AFSr13–gwm427 was deduced from the recombination data. Markers dupw167 and AFSr13 were validated on 21 durum wheat genotypes. Combination of dupw167 and AFSr13 would facilitate marker-assisted selection of Sr13 in segregating populations. At the hexaploid level, only gwm427 showed polymorphism and differentiated the presence of Sr13 in 10 of the 15 backcross derivatives carrying Sr13 from their Sr13-lacking recurrent parents.

Cytogenetic and Yq microdeletion screening studies in infertile males

Infertility is defined as the inability of a sexually active couple to achieve pregnancy despite unprotected intercourse for a period of greater than 12 months. Infertility affects 15% of the infertile couples and in about half of them, male factor is responsible. It is due to low number, abnormal or immobile sperm production, or blockages that prevent the delivery of sperm. Illnesses, injuries, chronic health problems, lifestyle choices and other factors can also play a role. A total of 130 infertile males (43 azoospermic, 31 asthenospermic, 7 cryptozoospermic, 20 oligoasthenozoospermic, 29 normozoospermic) were analyzed for the presence of chromosomal abnormalities and Y chromosome classical microdeletions in the azoospermia factor (AZF) regions. Multiplex PCR amplification of genomic DNA was performed using two STS markers for each of the three non-overlapping regions, AZFa (sY84, sY86), AZFb (sY127, sY134) and AZFc (sY254, sY255) following standard guidelines. The markers ZFX/ZFY and sY14 (SRY) were included as internal controls. Two individuals showed Klinefelter syndrome (47,XXY) and three cases exhibited reciprocal translocations involving autosomes - t(3;17)(q26.2;p12), t(1;11)(p22.3;p13) and t(1;10)(q25;q24). Polymorphisms involving the heterochromatic regions of Y and the acrocentric chromosomes were seen in seven patients. The rest of the individuals exhibited a normal chromosomal pattern. The occurrence of chromosomal abnormalities was confined to only the azoospermia category which could reflect the small sample size. A deletion involving all the three AZF regions was detected in one individual while another showed a deletion in AZFb (partial) and AZFc regions. These results indicate a distinctly lower frequency of Y chromosomal microdeletions in the selected group of men with idiopathic infertility. The study needs to be extended using more primer sets defining these loci which play a major role in spermatogenesis.

Isolation and characterization of a wheat – Psathyrostachys huashanica ‘Keng’ 3Ns disomic addition line with resistance to stripe rust

We isolated a wheat germplasm line, 22-2, which was derived from common wheat (Triticum aestivum '7182') and Psathyrostachys huashanica 'Keng' (2n = 2x = 14, NsNs). Genomic composition and homoeologous relationships of 22-2 was analyzed using cytology, genomic in situ hybridization (GISH), EST-SSR, and EST-STS to characterize the alien chromatin in the transfer line. The cytological investigations showed that the chromosome number and configuration were 2n = 44 = 22 II. Mitotic and meiotic GISH using P. huashanica genomic DNA as the probe indicated that 22-2 contained a pair of P. huashanica chromosomes. The genomic affinities of the introduced P. huashanica chromosomes were determined by EST-SSR and EST-STS using multiple-loci markers from seven wheat homoeologous groups between the parents and addition line. One EST-SSR and 17 EST-STS markers, which were located on the homoeologous group 3 chromosomes of wheat, amplified polymorphic bands in 22-2 that were unique to P. huashanica. Thus, these markers suggested that the introduced Ns chromosome pair belonged to homoeologous group 3, so we designated 22-2 as a 3Ns disomic addition line. Based on disease reaction to mixed races (CYR31, CYR32, and Shuiyuan14) of stripe rust in the adult stages, 22-2 was found to have high resistance to stripe rust, which was possibly derived from its P. huashanica parent. Consequently, the new disomic addition line 22-2 could be a valuable donor source for wheat improvement depending on the excellent agronomic traits, especially, the introduction of novel disease resistance genes into wheat during breeding programs.

Conversion of a male-specific RAPD marker into an STS marker in Asparagus officinalis L.

Garden asparagus (Asparagus officinalis L.) is an economically important plant. This species is dioecious, and male plants are considered to be more desirable than females due to their higher yields. To reduce the time required for asparagus breeding, molecular marker techniques have been employed to identify sex-linked DNA markers. In the present study, we converted the male-specific random amplified polymorphic DNA marker T35R54-1600 into a sequence tagged site marker. We cloned a male-specific DNA fragment amplified with the T35R54 primer and determined the sequence of the fragment. The size of T35R54-1600 is 1,586 bp, and this fragment is not homologous to known sex-linked BAC sequences, indicating that this fragment is a new sex-linked region. Within this fragment, we designed the primer pair ‘MSSTS710’ to amplify a 710 bp region. This marker could be used to identify the sex of eight cultivars of A. officinalis: ‘Mary Washington 500W’, ‘UC157’, ‘Harumachi Green’, ‘Super Welcome’, ‘F4’, ‘Pacific 2000’, ‘F2’ and ‘Backlim’. We also analyzed the applicability of this marker to two dioecious Asparagus species, A. schoberioides and A. kiusianus, which are cross-compatible with A. officinalis. Although male-specific DNA fragments of two dioecious Asparagus species, A. schoberioides and A. kiusianus, were generated using the existing male-specific marker Asp1T7sp, no amplicon was obtained using the MSSTS710 marker. Since MSSTS710 can be employed for sex identification only in A. officinalis and not in closely related Asparagus species, the DNA region around the MSSTS710 marker must be variable among Asparagus species.

Study on the components of tolerance and identification of resistance gene Lr34 with molecular tool to the wheat brown rust disease (Puccinia triticina) in the promising lines

Brown rust disease is one the most important fungal disease of wheat in Iran. Using of resistance cultivars is the best control method of disease. In this study, genetic resistance of 82 promising lines was evaluated in green house condition. Their resistance components included Infection Type, Latent Period, Postule Size and Postule Density in the randomised complete block design with two replications. In the molecular level for lines with corresponding adult plant resistance Lr34 gene, PCR process by means of STS marker and the primer Lr34/Yr18 was done. The results of variance analysis obtained implicated a significant difference in the 1% probable. The minimum value detected as −0.88 to the Gorgan isolate and the maximum value for −0.94 to Ahwaz isolate. In the cluster analysis related to two isolates, lines were divided to four groups composed. The presence of gene Lr34 was proved into the two genotypes “ER-S8–90” and “ER-N3–90”.