Morpho-molecular diversity study of rice cultivars in Bangladesh

Rice is one of the frontline cereals in the world and the major cultivated crop in Bangladesh. A total of eleven simple sequence repeats (SSRs) and thirteen sequence-tagged site (STS) markers were used to characterize twenty-four rice cultivars in Bangladesh. Twenty-four markers generated 60 alleles with 2.5 alleles per locus. The average polymorphism information content (PIC) value was 0.40, while the mean value of heterozygosity, gene diversity, and major allele frequency were recorded as 0.10, 0.48 and 0.62, respectively. However, the SSR markers showed more specificity and a higher discrimination power than the STS markers. The cluster analysis displayed four major clusters with a genetic similarity coefficient value of 0.73. The morphological analyses of the grain identified that Binadhan-20 and BRRI dhan34 had the longest and the shortest seed size, respectively, with a variable correlation between the seed length, width and length/width ratio. The phenol reaction test distinguished seven cultivars as japonica and seventeen cultivars as indica or an intermediate type. All these results regarding the phenotypic data and marker information will be useful for parental selection in modern rice breeding programmes.

Development of RNA-seq-based molecular markers for characterizing Thinopyrum bessarabicum and Secale introgressions in wheat

Sequence-based markers have added a new dimension in the efficiency of identifying alien introgressions in wheat. Expressed sequence tag-sequence tagged sites (EST-STS) markers have proved useful in tracing alien chromatin. In this study, we report the development of Thinopyrum bessarabicum- and Secale anatolicum-specific EST-STS markers and their application in tracing respective alien chromatin introgressions in wheat. The parental lines, Chinese Spring (CS), ISR991.1 (CS/Th. bessarabicum amphidiploid), and ISR1049.2 (CS/Secale anatolicum amphidiploid), were used as core experimental materials. Using comparative analysis of RNA-Seq data, 10 903 and 10 660 candidate sequences specific to Th. bessarabicum and S. anatolicum, respectively, were assembled and identified. To validate the genome specificity of these candidate sequences, 68 and 64 EST-STS markers were developed from randomly selected candidate sequences of Th. bessarabicum and S. anatolicum, respectively, and tested on sets of alien addition lines. Fifty-five and 53 markers for Th. bessarabicum and S. anatolicum chromatin, respectively, were assigned to chromosomal location(s), covering all seven chromosomes. Approximately 83% of S. anatolicum-specific markers were transferable to S. cereale. The genome-specific candidate sequences identified and the EST-STS markers developed will be valuable resources for exploitation of Th. bessarabicum and Secale species diversity in wheat and triticale breeding.

Mapping of the New Fertility Restorer Gene Rf-PET2 Close to Rf1 on Linkage Group 13 in Sunflower (Helianthus annuus L.)

The PET2-cytoplasm represents a well characterized new source of cytoplasmic male sterility (CMS) in sunflower. It is distinct from the PET1-cytoplasm, used worldwide for commercial hybrid breeding, although it was, as PET1, derived from an interspecific cross between Helianthus. petiolaris and H. annuus. Fertility restoration is essential for the use of CMS PET2 in sunflower hybrid breeding. Markers closely linked to the fertility restorer gene are needed to build up a pool of restorer lines. Fertility-restored F1-hybrids RHA 265(PET2) × IH-51 showed pollen viability of 98.2% ± 1.2, indicating a sporophytic mode of fertility restoration. Segregation analyses in the F2-population of the cross RHA 265(PET2) × IH-51 revealed that this cross segregated for one major restorer gene Rf-PET2. Bulked-segregant analyses investigating 256 amplified fragment length polymorphism (AFLP) primer combinations revealed a high degree of polymorphism in this cross. Using a subset of 24 AFLP markers, three sequence-tagged site (STS) markers and three microsatellite markers, Rf-PET2 could be mapped to the distal region of linkage group 13 between ORS1030 and ORS630. Three AFLP markers linked to Rf-PET2 were cloned and sequenced. Homology search against the sunflower genome sequence of HanXRQ v1r1 confirmed the physical location of Rf-PET2 close to the restorer gene Rf1 for CMS PET1. STS markers were mapped that can now be used for marker-assisted selection.

A 1Ns Disomic Addition from Psathyrostachys Huashanica Keng Confers Resistance to Powdery Mildew in Wheat

Powdery mildew is a fungal disease that threatens wheat production throughout the world. Breeding resistant cultivars is an effective way to reduce harm caused by powdery mildew. In this study, 35 wheat-Psathyrostachys huashanica-derived lines were developed by crossing common wheat and P. huashanica Keng (2n = 2x = 14, NsNs) using embryo culture. Resistance to powdery mildew in the derived lines was identified at the seedling and adult stages. Line H5-5-4-2 was selected with immunity to powdery mildew at both growth stages. The chromosome structure of this line was characterized by cytology, genomic in situ hybridization (GISH), and expressed sequence tag-sequence-tagged site (EST-STS) analysis. The chromosome configuration was 2n = 44 = 22II. Two P. huashanica chromosomes with strong hybridization signals were detected by GISH analysis. Among 83 EST-STS markers that covered all seven homologous groups in wheat, three pairs of STS markers, BE497584, BF202643, and BG262410, located in wheat homologous group 1 amplified clear specific bands related to P. huashanica. The results indicated that resistant line H5-5-4-2 was a wheat-P. huashanica 1Ns disomic addition line.

Study of Y-Chromosome Microdeletions in Azoospermic Infertile Males using Multiplex PCR Analysis

The infertility affects about 15% of couples and male factors being responsible about 40-50%. In male infertility, genetic abnormalities of Y chromosome play crucial role in spermatogenesis defect. Y chromosome q arm having Azoospermia factor region (AZFa, AZFb, and AZFc) are most important for spermatogenesis. Here, we investigated the frequencies of Y-chromosome microdeletions using three sets of multiplex PCR in idiopathic cases of azoospermia. We studied a total of 110 infertile male with non-obstructive azoospermia subjects & 50 fertile control subjects. All DNA samples were used for Y chromosome microdeletions analysis by using 11 STS markers in three different multiplex PCR of AZF regions. Out of 110 infertile azoospermic males, 14 (12.72%) infertile males showed partial deletion of AZF regions using three sets of multiplex PCR group. In the AZF microdeletions of infertile males, individually AZFc region was the most deletions sites (10%) followed by AZFb (6.36%) and AZFa (1.81%). The sites and sizes of microdeletions differ in all infertile azoospermic males who showed at least two or more STS markers microdeletions. The frequency of Y chromosome microdeletions in our azoospermic infertile males is 12.72%. We conclude that Y chromosome microdeletions frequency in azoospermic infertile males is higher than other infertile group due to severe impairment in spermatogenesis. Multiplex PCR screening of microdeletions is very useful and time saving technique when used more number of STS markers. It will be great help to infertility clinics for genetic counseling and assisted reproduction.

Development and Validation of High-Resolution Melting Markers Derived from Rysto STS Markers for High-Throughput Marker-Assisted Selection of Potato Carrying Rysto

Sequence analysis of the chromosome region harboring the sequence-tagged site (STS) markers YES3-3A and YES3-3B for Rysto, a gene responsible for extreme resistance to Potato virus Y (PVY) in potato, was performed in tetraploid potato ‘Barbara’ (Rrrr) and ‘AC Chaleur’ (rrrr) as well as their progeny selections. Three and two sequence variants were identified in Barbara resistant (R) selections and AC Chaleur susceptible (S) selections, respectively. Further analysis indicates that the variant with a 21-nucleotide (nt) deletion is likely the chromosome copy harboring the STS markers. Two primer pairs, one targeting the region containing a 20-nt deletion and the other targeting the region anchoring the YES3-3A reverse primer, were designed. As anticipated, pair one produced two visible fragments in Barbara-R bulk and one visible fragment in AC Chaleur-S bulk; pair two produced one visible fragment in all samples. When subjected to high-resolution melting (HRM) analysis, two distinct melting profiles for R and S samples were observed. Analysis of 147 progeny of Barbara × AC Chaleur revealed 72 and 75 progeny with R and S melting profiles, respectively, which was consistent with YES3-3A and YES3-3B assays and phenotyping analysis, thus demonstrating the potential of HRM profiles as novel molecular markers for Rysto. The efficacy of the newly developed HRM markers for high-throughput marker-assisted selection for Rysto-conferred resistance to PVY was validated further with three populations involving Barbara as the R parent.

Cytological and molecular analysis of wheat – Agropyron cristatum translocation lines with 6P chromosome fragments conferring superior agronomic traits in common wheat

Agropyron cristatum (2n = 4x = 28, PPPP) is a wild relative of common wheat and confers several desirable agronomic traits to wheat, such as high grain number per spike and enhanced resistance to certain diseases. Development of wheat – A. cristatum 6P translocation lines facilitates its utilization in wheat improvement. In this study, 26 wheat – A. cristatum 6P translocation lines were characterized by in situ hybridization (ISH) and 6P-specific sequence-tagged-site (STS) markers. These translocation lines carried different 6P chromosomal segments, which covered the whole 6P chromosome. FISH results showed that 15, 5, and 6 lines were translocated onto wheat A, B, and D genomes, respectively. Compared with the previous reports, a fine physical map of 6P chromosome was constructed, consisting of 31 chromosomal bins with 255 STS markers. Twelve translocation lines containing 6PS13∼14 chromosomal bins were highly resistant to leaf rust. Two lines showed high grain number per spike, and three lines displayed both enhanced grain number per spike and thousand-grain weight. Development of wheat – A. cristatum 6P translocation lines will not only provide novel wheat germplasm for wheat breeding but also be helpful to broaden the genetic basis of common wheat.