Sequence-tagged Site Research Articles

Mapping and KASP marker development for wheat curl mite resistance in “TAM 112” wheat using linkage and association analysis

Wheat curl mite (WCM, Aceria tosichella Keifer) and WCM-transmitted wheat streak mosaic virus (WSMV, genus Tritimovirus) are devastating production constraints for wheat in the US Great Plains. Breeding wheat cultivars with genetic resistance to WCM and WSMV is a viable and economically feasible way to reduce yield loss. The objectives of this study were to (a) identify tightly linked markers for WCM resistance in the wheat cultivar TAM 112 (PI 643143) using linkage and association analysis with the 90K Infinium iSelect SNP array and genotyping-by-sequencing, respectively and (b) develop and test kompetitive allele specific PCR (KASP) single-nucleotide polymorphisms (SNPs) for marker-assisted selection (MAS) of WCM resistance. We tested 124 F5:7 recombinant inbred lines (RILs) derived from the cross of TAM 112 and the WCM-susceptible cultivar TAM 111 (PI 631352). All lines were infested with a Texas WCM collection 2 (TWCMC2) that is virulent to resistance found on the wheat-rye 1AL.1RS translocation at the two-leaf stage and were rated for symptoms on the first and second week after infestation. Linkage maps were constructed with 4890 markers, including SNPs, simple sequence repeats (SSRs), and sequence-tagged site (STS) markers covering 21 chromosomes. A WCM resistance gene present in TAM 112 (CmcTAM112) was mapped onto chromosome arm 6DS. A genome-wide association study of wheat streak mosaic (WSM) symptoms from a separate experiment in Colorado showed significant marker-trait associations at the target regions on 6DS where CmcTAM112 was located, which demonstrated the effectiveness of this gene to reduce symptom severity. Four SNPs flanking CmcTAM112 were mapped within 3.6 cM in the biparental mapping population. We developed two KASP markers that are within 1.3 cM distal to CmcTAM112 and tested in diverse germplasm. These two markers can be used in MAS for improving WCM resistance in some wheat genetic backgrounds.

Identification of QTLs Associated withindica-japonicaDifferentiation-Related Traits in Rice (Oryza sativaL.)

Understanding the morphological and physiological characteristics which are attributable to determine indica and japonica subspecies of Asian rice (Oryza sativa L.) is important to conceive breeding strategy. However, information of quantitative trait loci (QTLs) associated with targeted traits related to subspecies domestication and differentiation-related traits remains limited. Two reciprocal F2 mapping populations generated from a cross between Ilpumbyeo (japonica) and Dasanbyeo (indica) were developed and used for constructing genetic maps to identify QTLs responsible for subspecies specific-related traits using 107 sequence-tagged site (STS) markers. A total of 26 main-effect QTLs for subspecies-related traits such as grain length, grain width, grain length-width ratio, apiculus hair length, and potassium chlorate resistance, were detected on seven chromosomes (1, 2, 3, 4, 5, 6, and 8). Of the detected QTLs, 14 QTLs corresponding to the previously reported QTLs suggesting the functional conservation of QTLs across populations and the remaining 12 QTLs appeared to be novel QTLs. A total of 29 significant epistatic QTLs were also detected in present study demonstrating an important genetic basis of epistatic interactions for complex subspecies-related traits. These results suggest that the subspecies-related traits and detected QTLs should be taken into consideration to incorporate with marker-assisted selection strategy in rice breeding program.

Assembly of a radish core collection for evaluation and preservation of genetic diversity

The radish is both an agronomically important root vegetable crop and a wild species of genus Raphanus in the Brassicaceae family. To date, at least four independent draft genome assemblies, marker development studies, and an analysis of radish evolution have been performed; however, there is no core collection of germplasm representing the genetic diversity in radish with minimum genetic redundancy. We report the assembly of a radish core collection consisting of 125 accessions. These accessions were selected by using 25 sequence-tagged site insertion/deletion markers to genotype 70 alleles in 886 accessions obtained from three germplasm banks in Korea, Japan, and Germany. An unweighted pair-group method with arithmetic mean (UPGMA) analysis of the genotyping data classified the accessions into 125 groups based on a Jaccard’s similarity coefficient of 0.72. One representative accession from each group was selected for a core collection that included 125 accessions, corresponding to 14.1% of the whole collection and including all 70 alleles. Principal coordinates analysis demonstrated that the genetic distribution within the core collection was similar to that of the whole collection, and 94% of the total variance within the whole collection could be explained using the analysis of molecular variance from the core collection. The UPGMA and STRUCTURE software analyses identified three major groups of accessions within the core collection, which were the Asian cultivated radish, the European cultivated radish, and the wild radish. The radish core collection developed in this study will be an important plant resource in future research and radish breeding.

The Prehistoric Indian Ayurvedic Rice Shashtika Is an Extant Early Domesticate With a Distinct Selection History.

Fully domesticated rice is considered to have emerged in India at approximately 2000 B.C., although its origin in India remains a contentious issue. The fast-growing 60-days rice strain described in the Vedic literature (1900–500 B.C.) and termed Shashtika (Sanskrit) or Njavara (Dravidian etymology) in Ayurveda texts including the seminal texts Charaka Samhita and Sushruta Samhita (circa 660–1000 B.C.) is a reliable extant strain among the numerous strains described in the Ayurveda literature. We here report the results of the phylogenetic analysis of Njavara accessions in relation to the cultivars belonging to the known ancestral sub-groups indica, japonica, aromatic, and aus in rice gene pool and the populations of the progenitor species Oryza rufipogon using genetic and gene genealogical methods. Based on neutral microsatellite markers, Njavara produced a major clade, which comprised of minor clades corresponding to the genotypic classes reported in Njavara germplasm, and was distinct from that were produced by the ancestral sub-groups. Further we performed a phylogenetic analysis using the combined sequence of 19 unlinked EST-based sequence tagged site (STS) loci with proven potential in inferring rice phylogeny. In the phylogenetic tree also the Njavara genotypic classes were clearly separated from the ancestral sub-groups. For most loci the genealogical analysis produced a high frequency central haplotype shared among most of the rice samples analyzed in the study including Njavara and a set of O. rufipogon accessions. The haplotypes sharing pattern with the progenitor O. rufipogon suggests a Central India–Southeast Asia origin for Njavara. Results signify that Njavara is genetically distinct in relation to the known ancestral sub-groups in rice. Further, from the phylogenetic features together with the reported morphological characteristics, it is likely that Njavara is an extant early domesticate in Indian rice gene pool, preserved in pure form over millennia by the traditional prudence in on-farm selection using 60-days maturity, because of its medicinal applications.

Genetic diversity and population structure among 133 elite genotypes of sugarcane (Saccharum spp.) for use as parents in sugarcane varietal improvement

A measure of genetic diversity of genotypes to be used as parents is imperative to use them prudently in crop improvement. In this study, genetic diversity and population structure of 133 sugarcane hybrid derivatives were quantified using 20 sequence-tagged microsatellite sites (STMS) primers. The number of alleles ranged from 9 to 27 with the average of 17.95 alleles per primer, while the polymorphism information content values of the primers ranged from 0.29 to 0.78. Cophenetic correlation coefficient value observed as 0.84 by STMS markers revealed that the cluster result was acceptable for the calculation of genetic similarity matrix. Principal component analysis showed that 133 genotypes fell in two groups, first and second components associated 8.34 and 3.22% with eigen values of 5.61 and 2.17, respectively. Similar trend was observed with principal coordinate analysis, wherein, the first and second component accounted to 8.34 and 3.22% with eigen values of 741.29 and 286.11. The similarity index values ranged from 0.50 to 0.87 for the possible 8778 combinations from 133 genotypes, of which 8069 combinations exhibited less/moderate genetic similarity indicating the availability of sufficient genetic diversity in the experimental material and hence their value in the genetic improvement of sugarcane. Dissimilarity analysis using DARwin of 133 genotypes could distinguish two major clusters and into five subclusters and the results matched with those of the population structure which also showed five subpopulations. The bigger group SP1 was predominantly comprised of clones developed at the main sugarcane-breeding place in India, located at Coimbatore. The subpopulation SP4 was formed largely with clones from research stations other than at Coimbatore and interspecific hybrids, while SP5 comprised of clones of early origin. These observations were similar to the radial tree based on the DARwin software in that 81.95% of the genotypes of each cluster were similar in the two analyses. The results thus showed that location and time of origin were two major factors that contributed to diversity. Based on analysis of molecular variance, subpopulations SP2 and SP4 were more variable from the rest. SP2 (comprising of Co 99008, Co 99006, Co 94012, Co 93023, CoC 671, Co 89034, Co 91003, Co 06022, Co 98017, Co 87044, Co 06018, Co 89003, Co 98014, and Co 86032) exhibited maximum genetic variation, the least gene flow, and the lowest heterozygosity value and would serve as the best group for utilization in genetic improvement. Graphical genotyping (GGT) image of each genotype was distinctly different, indicating the genetic uniqueness of sugarcane genotypes under study as revealed through STMS technology. A core set of 40 genotypes was identified using GGT 2.0 software program for the easiness of harnessing the available genetic diversity of 133 genotypes, through hybridization in sugarcane improvement programs.

Molecular mapping of leaf rust resistance gene LrL224 in Chinese wheat cultivar L224-3

Leaf rust, caused by Puccinia triticina, is a major wheat disease worldwide. The chinese wheat cultivar L224-3 showed high resistance to most of P. triticina pathotypes in the seedling and adult stage. Identifying and mapping the leaf rust resistance gene(s) in L224-3 is very useful for breeding leaf rust resistant wheat cultivars. In the present study, the wheat cultivar L224-3 and thirty-six lines with known leaf rust resistance genes were inoculated with 15 pathotypes at the seedling stage for gene postulation. A total of 144 F<sub>2:3</sub> lines from the cross L224-3 × Zhengzhou 5389 were inoculated with the pathotype FHBQ for leaf rust genetic analysis at the seedling stage. A total of 1276 SSR (simple sequence repeat) markers and the STS (sequence tagged-site) marker ω-secali/Glu-B3 were used to test the parents, resistant and susceptible bulks. The polymorphic markers were used to genotype the F<sub>2:3</sub> populations. L224-3 was highly resistant to all Lr26 avirulent pathotypes, showing the presence of Lr26 in L224-3. The presence of Lr26 in L224-3 was also confirmed by the molecular marker ω-secalin/Glu-B3. Due to resistance to some Lr26 virulent pathotypes, L224-3 may carry another resistance gene. Based on the genetic analysis using the pathotype FHBQ with virulence to Lr26 the resistance of L224-3 was controlled by a single dominant gene, tentatively designated LrL224. Four SSR markers (barc8, gwm582, wmc419, and wmc694) and one STS marker (ω-secali/Glu-B3) on 1B were closely linked to LrL224. The two flanking SSR loci were barc8 and gwm582, with the genetic distances of 4.3 and 4.6 cM, respectively. LrL224 was located on 1BL, and it showed different seedling reactions with other genes on 1B. Therefore LrL224 is likely to be a new leaf rust resistance gene.

Characterization of the p3-VvAGL11 Marker for Stenospermocarpic Seedlessness in Euvitis × Muscadinia Grape Hybrid Progenies

Stenospermocarpic seedlessness from Vitis vinifera L. is being introgressed into muscadine grape (Vitis rotundifolia Michx.) germplasm through the use of a cross-fertile hybrid of the two species. Recently, a sequence-tagged site (STS) molecular marker, p3_VvAGL11, has been developed which enables detection of the dominant allele controlling stenospermocarpic seedlessness in V. vinifera. This marker was evaluated in six Euvitis Planch. × Muscadinia Planch. hybrid progenies to determine its association with seedlessness in this material. The presence of the 214-bp seedlessness-associated p3_VvAGL11 allele in seedling vines resulted in a nearly 3-fold reduction in mean seed fresh weight (MSFW) and significantly reduced mean seed weight per berry (MSWB), percent berry weight composed of seed (BWCS), and mean berry weight (MBW). When the lack of lignified seed was used as the determinant of seedlessness, the p3_VvAGL11 marker was able to correctly judge seedlessness in ≈85% of the progeny. Analysis of seedlessness in the progenies was hampered by poor vigor and fruiting ability of the hybrid seedlings. The p3_VvAGL11 marker shows potential to speed the introduction of the stenospermocarpic seedlessness into Muscadinia germplasm by identifying seedless progeny at the seedling stage.

High resolution melting (HRM)-based STMS marker analysis for rapid identification of radiation induced mutants of sugarcane

Molecular marker based selection has become the method of choice to find undiscoverable natural variations and induced mutations in plants. Routine gel based molecular marker screening procedures involve complex steps which require considerable investment of time, money and efforts. Most often, genomic configuration of a plant species complicates the ultimate output of such screening programme. Therefore, a reliable and rapid high-throughput method is required to facilitate ease in identification of variants in a large number of samples. Here we demonstrate the application of a high-resolution melting (HRM) method to detect gamma radiation induced mutants of sugarcane (Saccharum spp.) which is a complex aneu-polyploidy plant. We have screened nine distinct mutants using sequence-tagged microsatellite site (STMS) markers and the analyses based on gel electrophoresis and HRM curve were compared. All the six STMS microsatellite markers coupled with HRM analysis were found highly informative and generated a unique melting curve for each of the marker tested. Of these, two markers NKSCSSR 22 and NKSCSSR 42 exhibited differential melting curve among the mutants and these results were well correlated with the gel based banding pattern. Taken together these results highlighted potential of HRM based marker screening and demonstrated reliability and robustness in detecting induced mutations in a complex polyploidy plant like sugarcane.

Dynamic quantitative trait loci (QTL) for plant height predict biomass yield in hybrid rye (Secale cereale L.)

The biomethane production from agricultural biomass will be a key component of the future energy mix. Rye (Secale cereale L.) is ideally suited for this purpose due to its high adaptability to a wide range of soils, weather and cropping conditions. Its high biomass yield is achieved with the lowest input of fertilizer and pesticides of all small-grain cereals. For the selection of cultivars with high kernel and biomass potential in the same breeding program, a prediction of biomass yield by indirect traits would increase the selection progress. To analyze quantitative-trait loci (QTL) for plant height in three developmental stages (BBCH 32, BBCH 51–55, BBCH 73) and biomass yield, 258 experimental rye hybrids were grown at three and four locations in Germany in 2011 and 2012, respectively. A genetic linkage map was constructed with 911 molecular markers. The QTL analysis identified a total of 18 QTL for plant height at the three assessment dates and of three QTL for dry matter yield at milk ripening. A major QTL on chromosome 2R explained 46, 12, and 25% of the total genetic variance for plant height at BBCH 51–55, BBCH 73, and for biomass yield, respectively. Furthermore, two large-effect QTL for plant height in two growth stages and dry matter yield were identified on chromosomes 3R and 5R. For eleven of the identified QTL in rye the respective QTL/genes on syntenic rice chromosomes were detected that could serve as candidate genes in follow-up experiments. A set of sequence-tagged site (STS) markers flanking the three QTL alleles for dry matter yield (QDmy-2R, QDmy-3R, and QDmy-5R) enables to the achievement of an indirect selection gain for biomass yield. This marker-assisted strategy allows for the screening of large hybrid rye populations already at early developmental stages without the resource-demanding harvest for biomass yield.

Contrasting patterns of variation in weedy traits and unique crop features in divergent populations of US weedy rice (Oryza sativa sp.) in Arkansas and California.

Weed evolution from crops involves changes in key traits, but it is unclear how genetic and phenotypic variation contribute to weed diversification and productivity. Weedy rice is a conspecific weed of rice (Oryza sativa) worldwide. We used principal component analysis and hierarchical clustering to understand how morphologically and evolutionarily distinct US weedy rice populations persist in rice fields in different locations under contrasting management regimes. Further, we used a representative subset of 15 sequence-tagged site fragments of expressed genes from global Oryza to assess genome-wide sequence variation among populations. Crop hull color and crop-overlapping maturity dates plus awns, seed (panicle) shattering (> 50%), pigmented pericarp and stature variation (30.2% of total phenotypic variance) characterize genetically less diverse California weedy rice. By contrast, wild-like hull color, seed shattering (> 50%) and stature differences (55.8% of total phenotypic variance) typify genetically diverse weedy rice ecotypes in Arkansas. Recent de-domestication of weedy species-such as in California weedy rice-can involve trait combinations indistinguishable from the crop. This underscores the need for strict seed certification with genetic monitoring and proactive field inspection to prevent proliferation of weedy plant types. In established populations, tillage practice may affect weed diversity and persistence over time. © 2017 Society of Chemical Industry.

Clinical, Hormonal, and Genetic Evaluation of Idiopathic Nonobstructive Azoospermia and Klinefelter Syndrome Patients

To investigate the clinical, hormonal, and genetic factors in infertile men with idiopathic nonobstructive azoospermia (NOA) or azoospermic Klinefelter syndrome (KFS), a total of 556 and 96 patients, respectively, were included in this study. All patient samples were analyzed cytogenetically. Serum reproductive hormone levels were measured. Microdeletions in the azoospermia factor (AZF) region of the Y chromosome were detected by multiplex PCR using 16 specific sequence-tagged sites. FSH and LH levels in both NOA and KFS patients were significantly higher than the normal range, and the testosterone level in KFS patients was significantly lower. Ninety-two (95.8%) of the KFS patients showed non-mosaic 47,XXY karyotypes and 47,XXY,inv(9)(p11.1q13); the other KFS patients had mosaic karyotypes of 47,XXY/46,XY, 47,XXY/46,XX, and 47,XXY/48,XXXY/46,XX. Among the 556 idiopathic NOA patients with normal karyotypes, 67 (12.05%) had microdeletions in the AZF region of the Y chromosome. Microdeletions were most frequently detected in the AZFc region, followed by AZFa, AZFb, AZFbc, and partial AZFc deletions. However, Y chromosome microdeletions were not found in any of the azoospermic KFS patients. In view of the hormonal and genetic abnormalities in infertile men with idiopathic NOA and with azoospermic KFS, genetic testing for karyotype, Y chromosome microdeletions, and hormonal parameters is advocated.

Characterization of Pm59, a novel powdery mildew resistance gene in Afghanistan wheat landrace PI 181356.

A new powdery mildew resistance gene, designated Pm59, was identified in Afghanistan wheat landrace PI 181356, and mapped in the terminal region of the long arm of chromosome 7A. Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is an important foliar disease of wheat worldwide. In the Great Plains of the USA, Bgt isolates virulent to widely used powdery mildew resistance genes, such as Pm3a, were previously identified. The objectives of this study were to characterize the powdery mildew resistance gene in Afghanistan landrace PI 181356, which exhibited high resistance to Bgt isolates collected in southern Great Plains, and identify molecular markers for marker-assisted selection. An F2 population and F2:3 lines derived from a cross between PI 181356 and OK1059060-126135-3 were used in this study. Genetic analysis indicated that PI 181356 carries a single dominant gene, designated Pm59, in the terminal region of the long arm of chromosome 7A. Pm59 was mapped to an interval between sequence tag site (STS) markers Xmag1759 and Xmag1714 with genetic distances of 0.4cM distal to Xmag1759 and 5.7cM proximal to Xmag1714. Physical mapping suggested that Pm59 is in the distal bin 7AL 0.99-1.00. Pm59 is a novel powdery mildew resistance gene, and confers resistance to Bgt isolates collected from the Great Plains and the state of Montana. Therefore, Pm59 can be used to breed powdery mildew-resistant cultivars in these regions. Xmag1759 is ideal for marker-assisted selection of Pm59 in wheat breeding.

Detection of Y Chromosome Microdeletions and Hormonal Profile Analysis of Infertile Men undergoing Assisted Reproductive Technologies.

Background Y chromosome deletions (YCDs) in azoospermia factor (AZF) region are associated with ab- normal spermatogenesis and may lead to azoospermia or severe oligozoospermia. Assisted reproductive tech- nologies (ART) by intracytoplasmic sperm injection (ICSI) and testicular sperm extraction (TESE) are com- monly required for infertility management of patients carrying YCDs. The aim of this study was to estimate the frequency of YCDs, to find the most frequent variant in infertile men candidate for ART and to compare YCD distribution with a control fertile group. The semen parameters, hormonal profiles and ART outcomes of the infertile group were studied. Materials and MethodsThis case-control study consisted of 97 oligozoospermic or non-obstructive azoospermic (NOA) infertile men, who had undergone ART, as the case group and 100 fertile men as the control group. DNA samples were extracted from blood samples taken from all 197 participants and YCDs were identified by multiplex polymerase chain reaction (PCR) of eight known sequence-tagged sites. The chi-square test was used to compare the mean values of hormone and sperm parameters between the two groups. P<0.05 was considered statistically significant.ResultsNo YCD was detected in the control group. However, 20 out of 97 (20.6%) infertile men had a YCD. AZFc, AZFbc and AZFabc deletions were detected in 15 (75%), four (20%) and one (5%) YCD-positive patients. No fer- tilization or clinical pregnancy was seen following ICSI in this sub-group with YCD. The mean level of FSH was significantly higher in the group with YCD (28.45 ± 22.2 vs. 4.8 ± 3.17 and 10.83 ± 7.23 in YCD-negative patients with and without clinical pregnancy respectively).ConclusionYCD is frequent among NOA men and YCD screening before ART and patient counseling is thus strongly recommended.

A boy with 46,X,+mar presenting gynecomastia and short stature.

A 15-year-old boy was referred due to gynecomastia and short stature. He was overweight and showed the knuckle-dimple sign on the left hand, a short fourth toe on the left foot, and male external genitalia with a small phallus. His levels of estradiol and follicle-stimulating hormone were increased, and his testosterone concentration was normal. Other hormonal tests were within the normal range. Radiographs showed short fourth and fifth metacarpals and fourth metatarsal bones. The karyotype was reported as 46,X,+mar, and the marker chromosome was shown to originate from the Y chromosome, which was identified by fluorescence in situ hybridization. Polymerase chain reaction and direct sequencing were used to clarify the deleted loci of the Y chromosome by making use of Y-specific sequence-tagged sites (STSs). The sex-determining region Y and centromere were verified, and there were microdeletions on the long arm of the Y chromosome. The azoospermia factor (AZF) b region was partially deleted, and AZFa and AZFc were completely deleted. Two STS probes of sY143 and the Y chromosome RNA recognition motif in AZFb showed positive signals corresponding to Yq11.223. The karyotype of the patient was interpreted as 46,X,der(Y)del(Y)(q11.21q11.222)del(Y)(q11.23qter). Herein, we report a rare case of a boy presenting with gynecomastia and short stature with 46, X, +mar, which originated from the Y chromosome, which was identified to have Yq microdeletions.

Development and implementation of a novel panel consisting 20 markers for the detection of genetic causes of male infertility.

Azoospermia factor (AZF) genes are involved in spermatogenesis. Deletions in the region of these genes have been recognised as a major genetic cause of infertility due to defects in spermatogenesis. Klinefelter syndrome (KS) is the other main cause of male infertility. This study was performed to establish a novel method for the detection of genetic causes of infertility in males and also to investigate the prevalence, extent and position of Y chromosome microdeletions in Iranian infertile men. We developed a newly designed panel of fluorescent multiplex-PCR method to amplify 20 markers (15 sequence-tagged sites (STSs) markers which are placed in the Y chromosome AZF region, 2 short tandem repeats (STRs) and 3 segmental duplications (SDs)). This multifunctional method is for the simultaneous detection of Y chromosome microdeletions and KS. Among 149 studied infertile men, one was detected to suffer from KS and seven (4.7%) were detected with the presence of one or more deleted STS loci. The main cause of infertility for the remaining patients would be nongenetic factors. This strategy is represented as a fast and accurate method to determine the frequencies of different AZF microdeletions which are suitable for use in clinical purposes.

Genetic Relationship of Stripe Rust Resistance Genes Yr34 and Yr48 in Wheat and Identification of Linked KASP Markers.

The Australian continent was free from wheat stripe rust caused by Puccinia striiformis f. sp. tritici until exotic incursions occurred in 1979 and 2002. The 2002 incursion enabled the identification of a new stripe rust resistance gene (Yr34) in the advanced breeding line WAWHT2046. In this study, we developed and validated markers closely linked with Yr34, which is located in the distal region in the long arm of chromosome 5A. Four kompetitive allele-specific polymerase chain reaction (KASP) and three sequence-tagged site (STS) markers derived from the International Wheat Genome Sequencing Consortium RefSeq v1.0 scaffold-77836 cosegregated with Yr34. Markers sun711, sun712, sun725, sunKASP_109, and sunKASP_112 were shown to be suitable for marker-assisted selection in a validation panel of 71 Australian spring wheat genotypes, with the exception of cultivar Orion that carried the Yr34-linked alleles for sunKASP_109 and sunKASP_112. Markers previously reported to be linked with adult plant stripe rust resistance gene Yr48 also cosegregated with Yr34. Wheat genotypes carrying Yr34 and Yr48 produced identical haplotypes for the Yr34-linked markers identified in this study and those previously reported to be linked with Yr48. Phenotypic testing of genotypes carrying Yr34 and Yr48 showed that both genes conferred similar seedling responses to pre-2002 and post-2002 P. striiformis f. sp. tritici pathotypes. Further testing of 600 F2 plants from a cross between WAWHT2046 and RIL143 (Yr48) with P. striiformis f. sp. tritici pathotype 134 E16A+Yr17+Yr27+ failed to reveal any susceptible segregants. Our results strongly suggest that Yr34 and Yr48 are the same gene, and that Yr48 should be considered a synonym of Yr34.

RNA-seq facilitates development of chromosome-specific markers and transfer of rye chromatin to wheat

Transcriptome shotgun sequencing (RNA-seq) provides an abundant resource for developing molecular markers specifically related to functional genes. In this study, transcriptomes of Chinese rye cultivar Jingzhouheimai (JZHM) challenged with powdery mildew pathogen Bgt (Blumeria graminis f. sp. tritici) were obtained and used to develop expressed sequence tag (EST)-based simple sequence repeat (SSR) and sequence-tagged site (STS) markers. A total of 866 primer sets for EST-SSRs and STSs were designed, from which we developed 401 rye-specific markers. The highest level of polymorphism was observed in EST-SSRs (56.73%) followed by STS2 (49.07%) designed via rye-specific contigs and STS1 (35.90%) primers designed from upregulated contigs. Genotyping with newly developed markers along with cytogenetic techniques allowed us to identify nine wheat alien chromosome lines from the cross of Zhoumai 18/Jinghui 1 (Jinghui 1 is an amphiploid of wheat landrace Huixianhong and JZHM) carrying rye chromosome 6R or 6R segments of different length, which permitted preliminary location of the powdery mildew resistance gene PmJZHM6RL and 12 specific markers to 6RL FL 0.51–1.0. 5R-specific markers and genomic in situ hybridization/fluorescence in situ hybridization detected MtA5RL and T5AS·5AL-5RL chromosomes among 41 F5 plants from the cross CS ph1bph1b/MA5R, and aberrations permitted the location of the hairy peduncle gene (Hp) and marker XLFZ3473 to the region 5RL FL 0.78–1.0 with another nine markers locating to 5RL 0.0–0.78. The chromosome-specific markers and chromosome aberrations developed in this study will facilitate the introgression of rye chromatin into wheat.

High frequency of microdeletion in TTY2 gene family in peripheral blood leukocytes of non-obstructive azoospermia patients.

About 10–15% of non-obstructive azoospermia (NOA) patients show AZFc microdeletion in their blood leukocytes. However, if AZF genes were involved in impaired spermatogenesis, a higher frequency of chromosomal microdeletions was expected. In this study the frequency of AZFc microdeletion was compared with TTY2 gene family, i.e., TTY2A2A and TTY2A12A in blood leukocytes of NOA patients and normal fertile control. In the present study 30 normal fertile individuals with mean age of 35.0 ± 6.0 and 30 NOA patients with mean age of 34.0 ± 7.0 were screened for microdeletion of TTY2L2A and TTY2L12A at Yq11 and Yp11 respectively and sequence-tagged site (STS) markers for AZFc gene using multiplex PCR technique. At the first step karyotyping was done for all subjects using standard G-banding technique to identify patients with normal karyotype as well as non-affected normal controls for molecular analysis.Results showed no AZFc microdeletion in normal and NAO patients whereas one TTY2L2A microdeletion in normal control (3.3%) and 4 in NOA (13.3%) was observed (p < 0.05). However our data indicated that 6 of 30 NOA patients (20%) showed TTY2L12A microdeletion whereas there was no observed microdeletion in normal control (p < 0.01).Results indicate that the studied genes might be involved in impaired spermatogenesis more effective than the routinely screened AZF genes in infertile men. Therefore, screening these genes along with AZF genes might be valuable for infertile patients. The reason why these genes are deleted from Y chromosome is not known but might be associated with genomic instability induced by environmental physico-chemical genotoxic agents.

Physical Localization of a Locus from Agropyron cristatum Conferring Resistance to Stripe Rust in Common Wheat.

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most destructive diseases of wheat (Triticum aestivum L.) worldwide. Agropyron cristatum (L.) Gaertn. (2n = 28, PPPP), one of the wild relatives of wheat, exhibits resistance to stripe rust. In this study, wheat-A. cristatum 6P disomic addition line 4844-12 also exhibited resistance to stripe rust. To identify the stripe rust resistance locus from A. cristatum 6P, ten translocation lines, five deletion lines and the BC2F2 and BC3F2 populations of two wheat-A. cristatum 6P whole-arm translocation lines were tested with a mixture of two races of Pst in two sites during 2015–2016 and 2016–2017, being genotyped with genomic in situ hybridization (GISH) and molecular markers. The result indicated that the locus conferring stripe rust resistance was located on the terminal 20% of 6P short arm’s length. Twenty-nine 6P-specific sequence-tagged-site (STS) markers mapped on the resistance locus have been acquired, which will be helpful for the fine mapping of the stripe rust resistance locus. The stripe rust-resistant translocation lines were found to carry some favorable agronomic traits, which could facilitate their use in wheat improvement. Collectively, the stripe rust resistance locus from A. cristatum 6P could be a novel resistance source and the screened stripe rust-resistant materials will be valuable for wheat disease breeding.

Marker assisted introgression of bacterial blight resistant gene into submergence tolerance rice variety BRRI dhan52

BRRI dhan52 is a uniquire submergence tolerant rice variety containing prominant genetic background of BR11, a mega rainfed lowland rice (RLR) variety of Bangladesh, but is susceptible to bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo). The variety is considerably popular in the southern part of Bangladesh due to its high yield in flash flood condition and comparatvely medium slender grain. Molecular markers linked to BB resistance genes (Xa genes) and submergence QTL (SUB1) were utilized in a marker-aided selection program to develop elite breeding lines with broad-spectrum resistance to bacterial blight. Sequence tagged site (STS) and simple sequence repeat (SSR) markers were essentially used to detect the genes for BB and submergence as well. In backcross generation, markers closely linked to Xa21 and SUB1 QTL were used to select desirable plants possessing these resistance genes (foreground selection) and microsatellite markers polymorphic between donor and recurrent parent were also used to select plants that have maximum contribution from the recurrent parent genome (background selection). In BC1F1 generation, three best plants consiquently were selected from previously selected ten double heterozygous (Xa21 and SUB1 QTL) plants. The percentage of recipient genome recovery in the best plant 1, 2 and 3 were 78.7%, 75.83% and 75.4%, respectively. Eventually this work illustrates the successful application of marker-assisted breeding for introgression of bacterial blight resistant gene into a rice variety of Bangladesh.Bangladesh J. Agril. Res. 42(3): 403-411, September 2017