Combination Breeding and Marker-Assisted Selection to Develop Late Blight Resistant Potato Cultivars

(1) Background: Although resistance to pathogens and pests has been researched in many potato cultivars and breeding lines with DNA markers, there is scarce evidence as to the efficiency of the marker-assisted selection (MAS) for these traits when applied at the early stages of breeding. A goal of this study was to estimate the potential of affordable DNA markers to track resistance genes that are effective against the pathogen Phytophthora infestans (Rpi genes), as a practical breeding tool on a progeny of 68 clones derived from a cross between the cultivar Sudarynya and the hybrid 13/11-09. (2) Methods: this population was studied for four years to elucidate the distribution of late blight (LB) resistance and other agronomical desirable or simple to phenotype traits such as tuber and flower pigmentation, yield capacity and structure. LB resistance was phenotypically evaluated following natural and artificial infection and the presence/absence of nine Rpi genes was assessed with 11 sequence-characterized amplified region (SCAR) markers. To validate this analysis, the profile of Rpi genes in the 13/11-09 parent was established using diagnostic resistance gene enrichment sequencing (dRenSeq) as a gold standard. (3) Results: at the early stages of a breeding program, when screening the segregation of F1 offspring, MAS can halve the workload and selected SCAR markers for Rpi-genes provide useful tools.

Development of Molecular Markers Associated with Resistance to Gray Mold Disease in Onion (Allium cepa L.) through RAPD-PCR and Transcriptome Analysis

Onions (Allium cepa L.) are one of the most consumed vegetable crops worldwide and are damaged by several fungal diseases in the field or during storage. Gray mold disease caused by the necrotrophic pathogens Botrytis cinerea and Botrytis squamosa is a disease that reduces the productivity and storage life in onions. However, it is difficult to control gray mold disease in onions by using physical and chemical methods. Breeding resistant onions against gray mold disease can reduce the damage caused by pathogens, reduce the labor required for control, and reduce environmental pollution caused by fungicides. However, onions have a large genome size (16Gb), making them difficult to analyze, and have a biennial cycle, resulting in a very long breeding period. Therefore, in this study, markers were developed to shorten the onion breeding period. First, random amplified polymorphic DNA (RAPD) was performed to confirm the genetic relationship between the gray mold disease-resistant and -susceptible lines through a dendrogram. In addition, the sequence characterized amplified region (SCAR)-OPAN1 marker to select resistant lines was developed using a polymorphic RAPD fragment. Second, the RNA-seq of the gray mold-resistant and -susceptible onion lines were analyzed using NGS technology. Using the RNA-seq results and DEG and GO analyses were performed, and the variants, such as SNPs and indels, were analyzed to develop a selectable marker for the resistant line. This study developed the SNP-3 HRM marker for selecting gray mold disease-resistant lines by using the SNPs present in the aldo-keto reductase (AKR) gene with high expression levels in these lines. The SCAR-OPAN1 and SNP-3 HRM markers developed in this study could be used to select gray mold disease-resistant onions in breeding programs to reduce the damage caused by gray mold disease.

Discovery and Chromosomal Location a Highly Effective Oat Crown Rust Resistance Gene Pc50-5

Crown rust, caused by Puccinia coronata f. sp. avenae, is one of the most destructive fungal diseases of oat worldwide. Growing disease-resistant oat cultivars is the preferred method of preventing the spread of rust and potential epidemics. The object of the study was Pc50-5, a race-specific seedling crown rust resistant gene, highly effective at all growth stages, selected from the differential line Pc50 (Avena sterilis L. CW 486-1 × Pendek). A comparison of crown rust reaction as well as an allelism test showed the distinctiveness of Pc50-5, whereas the proportions of phenotypes in segregating populations derived from a cross with two crown rust-susceptible Polish oat cultivars, Kasztan × Pc50-5 and Bingo × Pc50-5, confirmed monogenic inheritance of the gene, indicating its usefulness in oat breeding programs. Effective gene introgression depends on reliable gene identification in the early stages of plant development; thus, the aim of the study was to develop molecular markers that are tightly linked to Pc50-5. Segregating populations of Kasztan × Pc50-5 were genotyped using DArTseq technology based on next-generation Illumina short-read sequencing. Markers associated with Pc50-5 were located on chromosome 6A of the current version of the oat reference genome (Avena sativa OT3098 v2, PepsiCo) in the region between 434,234,214 and 440,149,046 bp and subsequently converted to PCR-based SCAR (sequence-characterized amplified region) markers. Furthermore, 5426978_SCAR and 24031809_SCAR co-segregated with the Pc50-5 resistance allele and were mapped to the partial linkage group at 0.6 and 4.0 cM, respectively. The co-dominant 58163643_SCAR marker was the best diagnostic and it was located closest to Pc50-5 at 0.1 cM. The newly discovered, very strong monogenic crown rust resistance may be useful for oat improvement. DArTseq sequences converted into specific PCR markers will be a valuable tool for marker-assisted selection in breeding programs.

Combination Breeding and Marker-Assisted Selection to Develop Late Blight Resistant Potato Cultivars

(1) Background: Although resistance to pathogens and pests has been researched in many potato cultivars and breeding lines with DNA markers, there is scarce evidence as to the efficiency of the marker-assisted selection (MAS) for these traits when applied at the early stages of breeding. A goal of this study was to estimate the potential of affordable DNA markers to track Rpi disease resistance genes, that are effective against the pathogen Phytophthora infestans, as a practical breeding tool on a progeny of 68 clones derived from a cross between the cultivar Sudarynya and 13/11-09. (2) Methods: this population was studied for four years to elucidate the distribution of LB resistance and other agronomical desirable or simple to phenotype traits such as tuber and flower pigmentation, capacity and structure of yield. LB resistance was phenotypically determined through natural and artificial infection and the presence/absence of nine Rpi genes was assessed via 11 sequence-characterized amplified region (SCAR) markers. To aid this analysis, the profile of Rpi genes in the 13/11-09 parent was established using diagnostic resistance gene enrichment sequencing (dRenSeq) as a gold standard. (3) Results: at the early stages of a breeding program, MAS can halve the workload when screening the segregation of F1 offspring and selected SCAR markers for Rpi-genes provide useful tools.

Distribution of Puccinia striiformis f. sp. tritici races and virulence in wheat growing regions of Kenya from 1970 TO 2014

Stripe rust, caused by the fungal pathogen Puccinia striiformis f. sp. tritici (Pst), is a major threat to wheat (Triticum spp.) production worldwide. The objective of this study was to determine the virulence of Pst races prevalent in the main wheat growing regions of Kenya, which includes Mt. Kenya, Eastern Kenya, and the Rift Valley (Central, Southern, and Northern Rift). Fifty Pst isolates collected from 1970 to 1992 and from 2009 to 2014 were virulence phenotyped using stripe rust differential sets, and 45 isolates were genotyped with sequence characterized amplified region (SCAR) markers to differentiate among the isolates and identify aggressive strains PstS1 and PstS2. Virulence corresponding to stripe rust resistance genes Yr1, Yr2, Yr3, Yr6, Yr7, Yr8, Yr9, Yr17, Yr25, Yr27 and the seedling resistance in genotype Avocet S were detected. Ten races were detected in the Pst samples obtained from 1970 to 1992, and three additional races were detected from 2009 to 2014, with a single race being detected in both periods. The SCAR markers detected both Pst1 and Pst2 strains in the collection. Increasing Pst virulence was found in the Kenyan Pst population, and that diverse Pst race groups dominated different wheat growing regions. Moreover, recent Pst races in east Africa indicated possible migration of some race groups into Kenya from other regions. This study is important in understanding Pst evolution and virulence diversity and useful in breeding wheat cultivars with effective resistance to stripe rust. Keywords: pathogenicity, Puccinia f. sp. tritici stripe (yellow) rust, Triticum aestivum

Marker-Assisted Pyramiding of Multiple Disease Resistance Genes in Coffee Genotypes (Coffea arabica)

The use of resistant cultivars is the most effective strategy for controlling coffee leaf rust caused by the fungus Hemileia vastatrix. To assist the development of such cultivars, amplified fragment-length polymorphism (AFLP) markers linked to two loci of coffee resistance to races I and II as well as pathotype 001 of H. vastatrix were converted to sequence-characterized amplified region (SCAR) and cleaved amplified polymorphic site (CAPS) markers. In total, 2 SCAR markers and 1 CAPS marker were validated in resistant and susceptible parents as well as in 247 individuals from the F2 population. The efficiency of these markers for marker-assisted selection (MAS) was evaluated in F2:3 and backcross (BCrs2) populations genotyped with the developed markers and phenotyped with race II of H. vastatrix. The markers showed 90% efficiency in MAS. Therefore, the developed markers, together with molecular markers associated with other rust resistance genes, were used for F3:4 and BCrs3 coffee selection. The selected plants were analyzed using two markers associated with coffee berry disease (CBD) resistance, aiming for preventive breeding. MAS of F3:4 and BCrs3 individuals with all resistance loci was feasible. Our phenotypic and genotypic approaches are useful for the development of coffee genotypes with multiple genes conferring resistance to coffee leaf rust and CBD.

Genetic Relationship And Species Identification of Dioscorea Polystachya Turcz. In Yams Determined By ISSR, ISAP, SRAP And SCAR Markers

Dioscorea polystachya Turcz. is the authentic herb Yam in Chinese traditional medicine. In the medicine market, some relative species are misused as “Yams”, such as D. alata L., D. exalata C.T. Ting & M.C. Chang, D. persimili Prain et Burkill, D. fordii Prain et Burkill and D. japonica Thunb. In order to estimate the genetic relationships among those species and develop a rapid and accurate method for D. polystachya identification, Inter Simple Sequence Repeat (ISSR), Intron Sequence Amplified Polymorphism (ISAP), Sequence-Related Amplified Polymorphism (SRAP) and Sequence Characterized Amplified Region (SCAR) were conducted in this study. Combining ISSR, ISAP and SRAP markers, a high degree of variation among the Dioscorea species was detected. Moreover, a relatively high gene flow (Nm = 0.3293) in those species was observed. The main reason for this phenomenon might be there were genepools exchanging in the wild materials. The phylogenetic tree and genetic similarity results showed D. japonica had the closest genetic relationship with D. polystachya. Considering the lowest inter-species variation observed in D. polystachya and the highest genetic divergence within D. japonica, we suggested the taxonomy of one species complex D. polystachya-D. japonica for breeding and conservation purpose. The phylogeny data in this study indicated D. alata and D. persimilis should be independent species respectively instead of cultigens. Furthermore, a SCAR marker was developed and could be used to discriminate the authentic herb D. polystachya from other confused Dioscorea species. Overall, the results are useful as guidelines for breeding programs and conservation actions.

Development, Identification, and Application of a Germplasm Specific SCAR Marker for Dendrobium officinale Kimura et Migo

The stems of Dendrobium officinale have been used as a rare and valuable Chinese tonic medicine, known as “Tiepi Fengdou”, since the Qing dynasty. Because of the increased market demand and continued exploitation of this plant, the reserves of wild D. officinale resources have been depleted, and D. officinale products on the market are being increasingly adulterated. Such changes have strongly affected the sustainable utilization of this valuable medicinal plant resource and the development of related industries. In this study, a species-specific DNA marker was developed for the rapid and accurate authentication of D. officinale. In total, 36 start codon-targeted (SCoT) polymorphism primers were screened in 36 definite Dendrobium species, and a distinct species-specific DNA amplicon (SCoT13-215) for D. officinale was obtained. After the sequence was cloned and sequenced, a sequence-characterized amplified region marker was developed (named SHF/SHR) and validated through PCR amplification of all 38 Dendrobium samples. The marker’s specificity for D. officinale was confirmed through the consistent amplification of a clear 197-bp band. This SCAR marker can be used to rapidly, effectively, and reliably identify D. officinale among various Dendrobium species and may play an important role in ensuring the quality of medicinal preparations and protecting the germplasm of this important medicinal species.

Production and Molecular Identification of Interspecific Hybrids between Phaius mishmensis (Lindl. and Paxton) Rchb. f. and Phaius tankervilliae (Banks) Blume

This study aimed at assessing the hybridization feasibility and evaluating genetic fidelity of the hybrid seedlings originated from Phaius mishmensis (Lindl. and Paxton) Rchb. f. and P. tankervilliae (Banks) Blume. Intra- and interspecific hybridization between Phaius mishmensis (Lindl. and Paxton) Rchb. f. and P. tankervilliae (Banks) Blume were examined to establish the primary hybrid, observe their cross ability and identify the F1 hybrids using sequence-characterized amplified region (SCAR) markers. Self-incompatibility and cross ability of P. mishmensis and P. tankervilliae were tested before starting the breeding program. Results showed that they were self-compatible orchids. The interspecific hybridization between P. mishmensis and P. tankervilliae was achieved with the highest pod setting (80%), seed germination percentage (94.8%) and the rate of protocorm development into mature seedlings (stage 6) (10.6%), but the smallest size of embryo with width 46.5 μm, length 67.3 μm was also observed when P. mishmensis was taken as the female parent. A comparative study on leaf morphology and anatomy of plantlets regenerated from intra- and interspecific hybrids of P. mishmensis and P. tankervilliae showed a transitional character to the parental species. Herein, the presence of interspecific hybrids between P. mishmensis and P. tankervilliae, as well as their reciprocal cross, was verified using Pmis524 SCAR markers developed by the decamer primer.