The development of resistant mungbean varieties is one of the most efficient strategies to control major diseases such as Cercospora leaf spot (CLS) and powdery mildew (PM). The objectives of this study were to pyramid a CLS resistance gene and two PM resistance genes from the donor parent D2 into a susceptible variety KING through marker-assisted backcrossing (MABC) and to evaluate their agronomic traits and disease resistance under field conditions. Five markers linked to the resistance genes were used for foreground selection, while two marker sets [Set A containing 15 polymorphic simple sequence repeat (SSR) and expressed sequence tag-SSR (EST-SSR) markers and Set B containing 34 polymorphic inter-simple sequence repeat (ISSR) loci] were also used for background selection. Two pyramided backcross (BC) lines, namely H3 and H4, were homozygous at all five marker loci when confirmed in BC4F4 and BC4F5 generations. Their recurrent parent genome (RPG) recovery ranged from 96.4 to 100.0%, depending on the marker sets. During field evaluation, a moderate to high level of CLS and PM resistance was observed in both BC lines compared to the susceptible recurrent parent KING. One of these BC lines (H3) had all agronomic traits similar or superior to the recurrent parent KING at all environments, and had a higher yield than KING (18.0–32.0%) under CLS and PM outbreaks. This line can be developed into a new resistant mungbean variety in Thailand in the future. These results substantiate the usefulness of MABC for transferring multiple resistance genes into an elite variety.
Commercial blueberry species of North America belong to the Vaccinium genus, section Cyanococcus. Phylogenetic relationships of 50 accessions of different ploidy levels within Cyanococcus were investigated using 249 expressed sequence tag-polymerase chain reaction markers and standard clustering methods. Of the commercial species, tetraploid V. corymbosum grouped most closely with the diploids, V. fuscatum and V. caesariense, followed by the diploid V. elliottii. Tetraploid V. angustifolium grouped with the diploids, V. boreale and V. myrtilloides. Hexaploid V. virgatum grouped most closely with the diploid V. tenellum, thus shedding light on the origins of these polyploid species.
Chrysanthemums (Chrysanthemum morifolium Ramat.) are ornamental flowers, which are famous worldwide. The mode of inheritance has great implications for the genetic analysis of polyploid species. However, genetic analysis of chrysanthemum has been hampered because of its controversial inheritance mode (disomic or hexasomic). To classify the inheritance mode of chrysanthemums, an analysis of three approaches was carried out in an F1 progeny of 192 offspring using 223 expressed sequence tag-simple sequence repeat (EST-SSR) markers. The analysis included segregation analysis, the ratio of simplex marker alleles linked in coupling to repulsion, as well as the transmission and segregation patterns of EST-SSR marker alleles. After segregation analysis, 204 marker alleles fit hexasomic inheritance and 150 marker alleles fit disomic inheritance, showing that marker alleles were inherited predominantly in a hexasomic manner. Furthermore, the results of the analysis of allele configuration and segregation behavior of five EST-SSR markers also suggested random pairing of chromosomes. Additionally, the ratio of simplex marker alleles linked in coupling to repulsion was 1:0, further supporting hexasomic inheritance. Therefore, it could be inferred that chrysanthemum is a complete or near-complete hexasome.
Ginger (Zingiber officinale Rosc.) is an economically important and valuable spice crop around the world. It is used as food, spice, condiment, and medicine. A considerable extent of genetic diversity in ginger occurs in the Western Ghats and North-Eastern India. However, genetic diversity studies at the molecular level in ginger is limited due to limited availability of genetic and genomic information. In the present study, for the first time, we have identified and validated expressed sequence tag (EST)-simple sequence repeat (SSR) markers from ginger. We obtained 16,790 EST-SSR loci from 78987 unigenes, and 4597 SSR loci in the predicted 76929 coding sequences from RNA-Seq assembled contigs of ginger through Illumina paired-end sequencing. Gene ontology results indicate that the unigenes with SSR loci participate in various biological processes such as metabolism, growth, and development in ginger. One hundred and twenty-five primer pairs were designed from unigenes and coding sequences. These primers were tested for PCR optimization, characterization, and amplification and identified 12 novel EST-SSR markers. Twelve flanking polymorphic EST-SSR primers were validated using 48 ginger genotypes representing North-Eastern India and different eco-geographical adaptations by PCR amplification and allele sizing through capillary electrophoresis. Twelve EST-SSR primers generated a total of 111 alleles with an average of 9.25 alleles per locus and allele sizes ranging between 115-189bp. This study implies that the SSR markers designed from transcriptome sequences provides ample EST-SSR resources, which are helpful for genetic diversity analysis of Zingiberaceae species and molecular verification of ginger genotypes.
Finger millet (Eleusine coracana (L.) Geartn.) is a self-pollinating amphidiploid crop cultivated with minimal input for food and feed, as well as a source of income for small-scale farmers. To efficiently assess its genetic diversity for conservation and use in breeding programs, polymorphic DNA markers that represent its complex tetraploid genome have to be developed and used. In this study, 13 new expressed sequence tag-derived simple sequence repeat (EST-SSR) markers were developed based on publicly available finger millet ESTs. Using 10 polymorphic SSR markers (3 genomic and 7 novel EST-derived), the genetic diversity of 55 landrace accessions and 5 cultivars of finger millet representing its major growing areas in Ethiopia was assessed. In total, 26 alleles were detected across the 10 loci, and the average observed number of alleles per locus was 5.6. The polymorphic information content (PIC) of the loci ranged from 0.045 (Elco-48) to 0.71 (UGEP-66). The level of genetic diversity did not differ much between the accessions with the mean gene diversity estimates ranging only from 0.44 (accession 216054) to 0.68 (accession 237443). Similarly, a narrow range of variation was recorded at the level of regional states ranging from 0.54 (Oromia) to 0.59 (Amhara and Tigray). Interestingly, the average gene diversity of the landrace accessions (0.57) was similar to that of the cultivars (0.58). The analysis of molecular variance (AMOVA) revealed significant genetic variation both within and among accessions. The variation among the accessions accounted for 18.8% of the total variation (FST = 0.19; P < 0.001). Similarly, significant genetic variation was obtained among the geographic regions, accounting for 6.9% of the total variation (P < 0.001). The results of the cluster, principal coordinate, and population structure analyses suggest a poor correlation between the genetic makeups of finger millet landrace populations and their geographic regions of origin, which in turn suggests strong gene flow between populations within and across geographic regions. This study contributed novel EST-SSR markers for their various applications, and those that were monomorphic should be tested in more diverse finger millet genetic resources.
The Australian finger lime is a unique citrus species that has gained importance due to its unique fruit characteristics and perceived tolerance to Huanglongbing (HLB), an often-fatal disease of citrus trees. In this study, we developed allotetraploid finger lime hybrids and cybrids by utilizing somatic cell fusion techniques to fuse diploid ‘OLL8’ sweet orange or ‘Page’ tangelo callus-derived protoplasts with finger lime (FL) mesophyll-derived protoplasts. Six somatic fusions were regenerated from the ‘OLL8’ + FL fusion, while three putative cybrids were regenerated from the ‘Page’ + FL fusion. Ploidy levels and nuclear-expressed sequence tag derived simple sequence repeat (EST-SSR) markers confirmed the somatic hybrid production, and mitochondrial DNA primer sets confirmed the cybrid nature. Several trees produced by the somatic fusion remained HLB negative even after 6 years of growth in an HLB-endemic environment. Pathogenesis related (PR) and other genes that are often upregulated in HLB-tolerant trees were also upregulated in our somatic fusions. These newly developed somatic fusions and cybrids could potentially be used as breeding parents to develop the next generation of improved HLB-tolerant rootstocks and scions.
The ornamental gourd Cucurbita pepo L. is a ubiquitous crop native to North America, exhibiting highly diverse fruit characteristics. Studying the genetic diversity of ornamental gourds can help identify and evaluate the curated germplasm resources, understand the phylogenetic relationships among them, and highlight ways in which the germplasm resources can be used to address gaps in the understanding. In this study, a set of 85 of 323 previously identified polymorphic expressed sequence tag-simple sequence repeat (EST-SSR) genetic markers were selected to evaluate the genetic relationships among 47 C. pepo accessions and one C. foetidissima accession. This collection consisted of accessions from the subspecies pepo, texana, and the hybrid texana × pepo. Our analyses yielded a total of 271 alleles, with an average of 3.2 alleles per genetic locus. The dendrogram construction, principal coordinate analyses, and genetic value calculation revealed several robust subclusters in the texana subspecies accessions. From these results, we propose five new distinct morphotypes based on our construction of a concise SSR fingerprint. Moreover, our study confirms that the fruit shape similarity among accessions is a fair reflection of genetic relatedness.