PCR-based Markers Research Articles

Towards the development of the PCR-based InDel markers associated with fruit skin quality traits in melon (Cucumis melo L.) using bulked segregant analysis

Melon's fruit skin quality is an agriculturally important and consumer-appealing trait. To identify molecular markers associated with fruit skin qualities, including reticulation and color, a total of 1200 Insertion/Deletion (InDel) markers randomly selected from the whole genome re-sequencing data of melon were combined with bulk segregant analysis to analyze a panel of melon genetic resources presenting reticulated- and smooth-skinned fruits as well as varying fruit skin colors. Four DNA bulks, including reticulated skin DNA bulk, smoothed skin DNA bulk, green skin DNA bulk, as well as white skin and light yellow skin DNA bulks, were developed. Four DNA pools were created by combining the DNA of 12 representative plants from each DNA bulk for polymorphism analysis, and 200 polymorphic InDel markers were selected. These polymorphic markers were used to characterize typical genetic bands within each DNA bulk. Two InDel markers, MC8-52 and MC4-7, were discovered to be related to fruit surface patterning (reticulated- and smooth-skinned fruits) and fruit color (green and white–light yellow fruits), respectively. These markers offer a high degree of detection accuracy. InDel marker MC8-52 on chromosome 8 achieved detection accuracies of 78.33% and 90.56%, whereas marker MC4-7 on chromosome 4 displayed detection accuracies of 92.92% and 83.35%, respectively. However, these markers were seen as a likely means of identifying strongly related markers for these traits. Hence, a high-resolution map of genomic regions carrying them is required for the development of highly linked markers for these traits.

Improving blast resistance of maintainer line DRR 9B by transferring broad spectrum resistance gene Pi2 by marker assisted selection in rice.

Hybrid rice technology offers great promise to further enhance rice production and productivity for global food security. Improving hybrid rice parental lines is the first step in developing heterotic rice hybrids. To improve resistance against blast disease, a maintainer line DRR 9B was fortified with a major broad-spectrum blast resistance gene Pi2 through marker-assisted selection. The rice blast caused by Magnaporthe oryzae is a major disease and can cause severe yield losses upto 100%. The NILs of Samba Mahsuri namely BA-23-11-89-12-168 possessing Pi2 was utilized as a donor parent. The PCR-based molecular marker tightly linked to Pi2 gene was used for the foreground selection at BC1F1 generation. The molecular marker tightly linked to the major fertility restorer gene Rf4 was used for negative selection (i.e., selection of plants possessing non fertility restoring alleles) at BC1F1 generation to identify maintainer lines. The positive plants with Rf4 gene were added to the restorer pool for restorer line development. At each stage, MAS for Pi2 coupled with stringent phenotypic selection for agro-morphological and grain quality traits were exercised. At BC1F3 generation, one hundred families were screened against blast disease at uniform blast nursery (UBN) and selected resistant lines were advanced to next generations. In the BC1F5 generation plants were subjected to agro-morphological evaluation for yield and yield-contributing traits. The selected plants at BC1F5 generation were crossed with DRR 9A to assess the maintainer ability of blast resistance lines and for further CMS line conversion for hybrid rice breeding for developing blast resistance rice hybrids.

An Economic Method to Identify Cultivars and Elite Lines in Radish (Raphanus sativus L.) for Small Seed Companies and Independent Breeders

Radish is a root vegetable crop, and many F1 cultivars have been developed by exploiting heterosis. Considerable time and resources are required to test the performance and combining ability of F1 cultivars and parental lines; therefore, economical methods to identify them are needed before field tests. This study was conducted to determine the minimum number of markers needed to identify cultivars and elite lines. PCR-based markers were applied to radish cultivars and elite lines and selected based on chromosomal location, polymorphic information content, and allele frequency. The power of selected marker sets to detect genetically similar cultivars or elite lines was confirmed using cultivars with high genetic similarity and elite lines with the same parents. Eleven markers successfully identified not only all 105 radish cultivars tested but also cultivars with high genetic similarity reported in previous studies. Fourteen markers, including the nine among 11 markers for cultivar identification, also successfully distinguished all 115 elite lines tested, including ones with the same parents and separated at F2–F6 generations. The markers developed for the identification of radish cultivars and elite lines require only 11 and 14 markers, respectively, and simple PCR and gel electrophoresis instruments. This method is very simple and can be easily applied to other crops.

The Efficacity Investigation for Some Markers Detecting Yellow Rust Resistance Genes in Bread Wheat Varieties

Yellow rust is (Puccinia striiformis Westend. f. sp. tritici) is among the supreme diseases causing serious losses in wheat production. The chemical fungicides are commonly used in this disease-fighting. However, chemical control is not economical and also causes environmental pollution. Therefore, the use of resistant wheat varieties in production has critical importance. The resistance against yellow rust disease is expressed with Yr genes. In the breeding studies, knowing which parents include resistance genes provides a great advantage in the development of new resistant varieties. This study aims to determine the efficiency of markers used to detect resistance genes against yellow rust disease. The efficiency of molecular markers (Xgwm582, RgaYr10a, Xgwm413, Xgwm11, Wmc44, Barc101, Cfa2149, Sun104, Xgwm273) that are identified for nine genes (Yr9, Yr10, Yr15, Yr26, Yr29, Yr36, Yr48, Yr51, and YrCH52) providing resistance against yellow rust disease was investigated using PCR method. Twenty bread wheat varieties were used as material. Resistance gene profiles determined using PCR-based molecular markers and data obtained from registration information and field resistance data in the literature were analysed comparatively. As a result of the analysis, the efficiency/productivity of the markers defined for different resistance genes in detecting the resistance gene profile of wheat varieties was determined. Moreover, resistance gene profiles of varieties that are known resistance states in the field and sensitive varieties were compared. Genes that are prominent in providing resistance and detected with markers were determined and the efficiency of these genes was evaluated according to their homozygous/heterozygous states. It was concluded that the efficacy of markers such as RgaYr10a, Xgwm413, Barc101, and Cfa2149, which gave positive results in all wheat varieties, was low.

\(\textit{Fusarium fujikuroi}\) WQF5 isolated from \(\textit{Cephalotaxus mannii }\)Hook.f. as a producer of antibacterial agent and paclitaxel

Cephalotaxus mannii Hook.f is a rare medicinal plant used for the treatment of leukemia, however, its fungal endophytes with antibacterial and anticancer properties have not been exploited yet. In this study, six fungal isolates were recovered from roots of C. mannii collected in Ha Giang province, Vietnam. Among them, ethyl acetate crude extract of strain WQF5 exhibited strong antibacterial activity against 5 tested pathogens with inhibition zones ranging from 13.0 ± 0.5 mm to 20.0 ± 0.4 mm. Sulforhodamine B assay showed that WQF5 extract possessed the most potent cytotoxic activity against lung cancer A549 and breast cancer MCF7 cell lines with IC50 values of 6.9 ± 0.7 µg/mL and 23.1 ± 1.9 µg/mL, respectively. PCR-based molecular marker screening revealed that the positive hits for essential genes encoding 10-deacetylbaccatin III-10-O-acetyltransferase (dbat) and C-13 phenylpropanoyl side chain-CoA acyltransferase (bapt) involved in paclitaxel production were found in the fungal isolate WQF5. In addition, isolate WQF5, identified as Fusarium fujikuroi by morphological and ITS analysis, also produced paclitaxel as shown by HPLC-DAD analysis. This is the first report of bioprospecting endophytic fungi isolated from C. mannii, in which the capability of producing paclitaxel of endophytic fungi was also proved for the first time. These findings addressed a potent candidate for paclitaxel production and provided excellent material for further investigations of how endophytic fungi from non-Taxus plant species synthesize paclitaxel.

Evaluation of Genetic Diversity in Dog Breeds Using Pedigree and Molecular Analysis: A Review

Domestic dogs are important for many economic and social reasons, and they have become a well-known model species for human disease. According to research, dog breeds exhibit significant levels of inbreeding and genetic diversity loss, decreasing the population’s ability to adapt in certain conditions, and indicating the need of conservation strategies. Before the development of molecular markers, pedigree information was used for genetic diversity management. In recent years, genomic tools are frequently applied for accurate estimation of genetic diversity and improved genetic conservation due to incomplete pedigrees and pedigree errors. The most frequently used molecular markers include PCR-based microsatellite markers (STRs) and DNA sequencing-based single-nucleotide polymorphism markers (SNP). The aim of this review was to highlight genetic diversity studies on dog breeds conducted using pedigree and molecular markers, as well as the importance of genetic diversity conservation in increasing the adaptability and survival of dog breed populations.

Integrated genomic approaches delineate a novel role of ROP1 ENHANCER1 in controlling seed protein content of chickpea.

Utilizing a combinatorial approach of quantitative trait locus (QTL)-Seq and candidate gene-based association mapping, the QTLs and genes responsible for seed protein content (SPC), a major quality trait in chickpea, were identified. Whole genome re-sequencing based QTL-Seq analysis of bulked recombinant inbred lines from a mapping population contrasting for SPC led to the identification of two QTLs [0.94 Mb on Linkage Group (LG)5 and 1.16 Mb on LG6] encompassing three SNPs, displaying the highest ΔSNP index. These highly significant SNPs and their associated genes were validated in 211 chickpea mini-core accessions varying in SPC, revealing a tightly associated marker affecting CaREN1 (ROP1 ENHANCER1) and explaining a phenotypic variation of 23%. This SNP was subsequently converted into a cost effective allele-specific PCR-based marker that could be utilized for rapid screening of SPC during marker assisted breeding. Furthermore, in planta functional validation via knockdown of CaREN1 transcripts led to significant reduction in SPC of chickpea. This decrease in seed protein is likely due to disruption in the formation of CaREN1 protein complexes comprising chaperones, phosphopeptide-binding proteins, and GTPases that mediate folding, transport and accumulation of seed storage proteins, as indicated through affinity purification-mass spectrometry. Taken together, our data will expedite tailoring of chickpea cultivars with augmented SPC.

Incorporation of Organic Growth Additives to Enhance In Vitro Tissue Culture for Producing Genetically Stable Plants.

The growing demand for native planting material in ecological restoration and rehabilitation for agro-silvo-pastoral ecosystems has resulted in a major global industry in their sourcing, multiplication, and sale. Plant tissue culture is used for producing high-quality, disease-free, and true-to-type plants at a fast rate. Micropropagation can help to meet the increasing demand for planting material and afforestation programs. However, in vitro plant propagation is an expensive technique compared to conventional methods using suckers, seeds, and cuttings. Therefore, adopting measures to lower production costs without compromising plant quality is essential. This can be achieved by improving the culture media composition. Incorporating organic growth additives can stimulate tissue growth and increase the number of shoots, leaves, and roots in culture media. Organic growth supplementation speeds up the formation and development of cultures and yields vigorous plants. Plant regeneration from meristems (shoot tips and axillary buds) is a reliable way to produce true-to-type plants compared with callus and somatic embryogenesis regeneration, but in vitro culture environments can be mutagenic. Therefore, detecting somaclonal variations at an early stage of development is considered crucial in propagating plants. The genetic stability of in vitro regenerated plants needs to be ascertained by using DNA-based molecular markers. This review aims to provide up-to-date research progress on incorporating organic growth additives to enhance in vitro tissue culture protocols and to emphasize the importance of using PCR-based molecular markers such as RAPD, ISSR, SSR, and SCoT. The review was assessed based on the peer-reviewed works published in scientific databases including Science Direct, Scopus, Springer, JSTOR, onlinelibrary, and Google Scholar.

Identification and Genome-Wide Association Analysis for Fusarium Crown Rot Resistance in Wheat.

Fusarium crown rot (FCR) is a fungal disease and severely decreases wheat production worldwide. Tibetan semiwild wheat, Yunnan hulled wheat, Xinjiang rice wheat, and Sichuan white wheat are four subspecies landraces endemic to western China and have rich genetic diversity in response to biotic and abiotic stresses. Here, a natural population, including 209 wheat accessions of four subspecies, was evaluated for FCR resistance. he genome-wide association study was performed using the wheat 55K single-nucleotide polymorphisms (SNPs). The results showed that the disease index (DI) ranged from 16.88 to 85.00, while six accessions showed moderate to high resistance (DI ≤ 30). Genome-wide association analysis identified 10 stable loci for FCR resistance on chromosomes 1B, 2A (5), 5A, 7A, 7B, and 7D. Four major loci-Qfcr.sicau.2A-1, Qfcr.sicau.2A-3, Qfcr.sicau.5A, and Qfcr.sicau.7D-explained 6.01 to 14.48, 9.76 to 13.11, 8.19 to 10.29, and 5.76 to 12.21% phenotypic variation, respectively. Quantitative trait loci (QTL) pyramiding analysis of these four major loci revealed that accessions with four resistance haplotypes could significantly decrease FCR severity by 9.35 to 31.61% compared with those without or with one to three resistance haplotypes. One kompetitive allele-specific PCR (KASP) marker each was successfully developed for Qfcr.sicau.2A-1 and Qfcr.sicau.7D. The KASP marker of Qfcr.sicau.2A-1 was used to genotype in an F6 recombinant inbred line population. The result showed that the lines carrying the resistance allele reduced FCR severity by 17.78%, demonstrating the importance of Qfcr.sicau.2A-1 in resistance breeding programs. Our findings provide valuable QTL and breeder-friendly PCR-based markers for applications in FCR resistance breeding programs. Our study also proved that gene pyramiding of major loci could enhance FCR resistance.

Distribution, cytotoxicity, and antioxidant activity of fungal endophytes isolated from Tsuga chinensis (Franch.) Pritz. in Ha Giang province, Vietnam

PurposeAn endangered Tsuga chinensis (Franch.) Pritz. is widely used as a natural medicinal herb in many countries, but little has been reported on its culturable endophytic fungi capable of producing secondary metabolites applied in modern medicine and pharmacy. The present study aimed to evaluate the distribution of fungal endophytes and their cytotoxic and antioxidant properties.MethodsThis study used the surface sterilization method to isolate endophytic fungi which were then identified using morphological characteristics and ITS sequence analysis. The antimicrobial and cytotoxic potentials of fungal ethyl acetate extracts were evaluated by the minimum inhibitory concentration (MIC) and sulforhodamine B (SRB) assays, respectively. Paclitaxel-producing fungi were primarily screened using PCR-based molecular markers. Additionally, biochemical assays were used to reveal the antioxidant potencies of selected strains.ResultsA total of sixteen endophytic fungi that belonged to 7 known and 1 unknown genera were isolated from T. chinensis. The greatest number of endophytes was found in leaves (50%), followed by stems (31.3%) and roots (18.7%). Out of 16 fungal strains, 33.3% of fungal extracts showed significant antimicrobial activities against at least 4 pathogens with inhibition zones ranging from 11.0 ± 0.4 to 25.8 ± 0.6 mm. The most prominent cytotoxicity against A549 and MCF7 cell lines (IC50 value < 92.4 μg/mL) was observed in Penicillium sp. SDF4, Penicillium sp. SDF5, Aspergillus sp. SDF8, and Aspergillus sp. SDF17. Out of three key genes (dbat, bapt, ts) involved in paclitaxel biosynthesis, strains SDF4, SDF8, and SDF17 gave one or two positive hits, holding the potential for producing the billion-dollar anticancer drug paclitaxel. Furthermore, four bioactive strains also displayed remarkable and wide-range antioxidant activity against DPPH, hydroxyl radical, and superoxide anion, which was in relation to the high content of flavonoids and polyphenols detected.ConclusionThe present study exploited for the first time fungal endophytes from T. chinensis as a promising source for the discovery of new bioactive compounds or leads for the new drug candidates.

Genetic Diversity and Population Structure of the Endangered Ulmusvillosa in Pakistan Revealed by DNA Barcode Markers

Ulmus villosa Brandis ex Gamble, an economically and ecologically important forest tree, is native to the western Himalayas of Pakistan. The long pressure imposed by unsustainable utilization and market demands has resulted in the rapid decline of the U. villosa population in the wild. To date, very limited information on the genomic diversity of U. villosa is available and this can tremendously limit our understanding of distribution and future conservation of U. villosa. Therefore, the current study aimed to assess genetic diversity within U. villosa wild populations of the Hazara Division using four barcoding markers (i.e., rbcL, matK XF + 5R, matK 390F + 1326R and trnH-psbA). A total of six high quality sequences were obtained with rbcL, four with matK XF + 5R, four with matK 390F + 1326R, and fourteen with trnH-psbA. The sequenced regions contained insertions and deletions as well as several SNPs across the length of sequences, and PCR-based markers may be developed from these novel SNPs markers. The phylogenetic results supported the polyphyletic origin of the genus Ulmus, and the data indicated that multiple speciation events may have led to the evolution of U. villosa in this region. For deeper understanding of the origin and evolution of U. villosa, sequencing of the complete nuclear and chloroplast genomes will be pivotal. The results herein provide novel insights into the genomic diversity as well as phylogenetic relationships of U. villosa with other species, and may facilitate both in situ and ex situ conservation efforts for this endangered species.

Cytogenetic and Molecular Marker Analyses of a Novel Wheat-Psathyrostachys huashanica 7Ns Disomic Addition Line with Powdery Mildew Resistance.

Powdery mildew caused by Blumeria graminis f. sp. tritici is a devastating disease that reduces wheat yield and quality worldwide. The exploration and utilization of new resistance genes from wild wheat relatives is the most effective strategy against this disease. Psathyrostachys huashanica Keng f. ex P. C. Kuo (2n = 2x = 14, NsNs) is an important tertiary gene donor with multiple valuable traits for wheat genetic improvement, especially disease resistance. In this study, we developed and identified a new wheat—P. huashanica disomic addition line, 18-1-5—derived from a cross between P. huashanica and common wheat lines Chinese Spring and CSph2b. Sequential genomic and multicolor fluorescence in situ hybridization analyses revealed that 18-1-5 harbored 21 pairs of wheat chromosomes plus a pair of alien Ns chromosomes. Non-denaturing fluorescence in situ hybridization and molecular marker analyses further demonstrated that the alien chromosomes were derived from chromosome 7Ns of P. huashanica. The assessment of powdery mildew response revealed that line 18-1-5 was highly resistant at the adult stage to powdery mildew pathogens prevalent in China. The evaluation of agronomic traits indicated that 18-1-5 had a significantly reduced plant height and an increased kernel length compared with its wheat parents. Using genotyping-by-sequencing technology, we developed 118 PCR-based markers specifically for chromosome 7Ns of P. huashanica and found that 26 of these markers could be used to distinguish the genomes of P. huashanica and other wheat-related species. Line 18-1-5 can therefore serve as a promising bridging parent for wheat disease resistance breeding. These markers should be conducive for the rapid, precise detection of P. huashanica chromosomes and chromosomal segments carrying Pm resistance gene(s) during marker-assisted breeding and for the investigation of genetic differences and phylogenetic relationships among diverse Ns genomes and other closely related ones.

Identification and Characterization of Wheat-Aegilops comosa 7M (7A) Disomic Substitution Lines with Stripe Rust and Powdery Mildew Resistance.

Aegilops comosa (MM, 2n = 2x = 14), an important diploid species from the wheat tertiary gene pool, contains many unique genes/traits of potential use for wheat breeding, such as disease resistance. In this study, three sister lines, NAL-32, NAL-33, and NAL-34, were identified from a wheat-A. comosa distant cross using fluorescence in situ hybridization, simple sequence repeat markers, and PCR-based unique gene markers combined with single nucleotide polymorphism (SNP) array analysis. Genetically, NAL-32 contained neither an alien nor translocation chromosome, whereas NAL-33 and NAL-34 had disomic 7M (7A) substitution chromosomes but differed in the absence or presence of the 1BL/1RS translocation chromosomes, respectively. The absence of 7A in NAL-33 and NAL-34 and the unusual 1B in the latter were verified by wheat 55K SNP arrays. The two 7M (7A) substitution lines had similar levels of resistance to stripe rust and powdery mildew, but better than that of NAL-32 and their common wheat parents, suggesting that the stripe rust and powdery mildew resistance of NAL-33 and NAL-34 were derived from the 7M of A.comosa. This research provides important bridge materials that can potentially be used for transferring stripe rust and powdery mildew resistance.

A dominant gene Ihrl1 is tightly linked to and inhibits the gene Ndhrl1 mediating nitrogen-dependent hypersensitive reaction-like phenotype in wheat.

Identification and mapping of an inhibitor of Ndhrl1 mediating nitrogen-dependent hypersensitive reaction-like phenotype in wheat. Hypersensitive reaction-like (HRL) traits are characteristic of spontaneous lesions including yellowish spots, brown spots or white-stripe that appeared randomly and dispersedly on all the leaves in the absence of plant pathogens. Our previous studies have shown that the wheat line P7001 showed an HRL trait at low nitrogen supply, and this trait was controlled by the gene Ndhrl1 (Nitrogen-dependent hypersensitive reaction-like 1). In order to investigate the robustness of the trait expression mediated by Ndhrl1 under different genetic backgrounds, seven genetic populations, with P7001 being the common female parent, were constructed and analyzed. F1 plants from six of the seven combinations showed HRL trait and Ndhrl1 segregated in a dominant way of HRL: non-HRL = 3:1 in the six populations (F2). Exceptionally, the F1 plants of P7001/Fielder combination showed non-HRL trait and HRL trait in the F2 population showed a contrasting recessive segregation ratio of HRL: non-HRL = 1:3, suggesting Fielder may have another HRL-related gene. Using 55K SNP array and PCR-based markers, the HRL-related gene in Fielder was mapped to an interval of 5.63-12.91Mb on the short arm of chromosome 2B with the flanking markers Yzu660R075552 and Yzu660F075941. A recombinant with genomic region of Fielder atNdhrl1 locus showingHRL trait demonstrated that Fielder also harbored Ndhrl1 same as P7001. Thus, Fielder carries a single dominant suppressor of Ndhrl1, designated as Ihrl1 (Inhibitor of hypersensitive reaction-like). Interestingly, Ihrl1 is tightly linked to Ndhrl1 and may be also involved in nitrogen metabolic and (or) signaling pathways.

The Sorting Nexin Genes ChSNX4 and ChSNX41 Are Required for Reproductive Development, Stress Adaption and Virulence in Cochliobolus heterostrophus.

Sorting nexins are a conserved protein family involved in many cellular processes in fungi, and the function of sorting nexin Snx4 (Atg24) and Snx41 (Atg20) in Cochliobolus heterostrophus was not clear. The ΔChsnx4 and ΔChsnx41 mutants were generated by a PCR-based marker method to determine the roles of Snx4 and Snx41 in reproductive development, stress adaption, and virulence in C. heterostrophus. Compared with the wild-type strain, the ΔChsnx4 and ΔChsnx41 mutants exhibited obvious changes in vegetative growth and in morphology of conidia. In addition, the conidiation, appressorium formation, and virulence of snx4 and snx41 mutants were dramatically reduced. Moreover, ΔChsnx4 and ΔChsnx41 mutants were more sensitive to oxidative stress (menadione and H2O2), cell wall integrity stress (Congo red and calcofluor white), fungicides, and isothiocyanates. All the phenotypes mentioned above were restored in complemented strains. In addition, ChSnx4 and ChSnx41 were proven to interact with each other through yeast two-hybrid. Taken together, these findings indicated that ChSNX4 and ChSNX41 were important for fungal growth, asexual development, stress adaption, and virulence in C. heterostrophus.

Development of PCR-based markers for the identification and detection of Lophodermella needle cast pathogens on Pinus contorta var. latifolia and P. flexilis

Morphological similarities and fastidious development of increasingly emerging fungal needle pathogens impede accurate disease diagnosis and early detection. This study analyzed the specificity and sensitivity of polymerase chain reaction (PCR)-based markers developed for emerging needle cast pathogens Lophodermella concolor and L. montivaga co-occurring on Pinus contorta var. latifolia, and Bifusella linearis and L. arcuata on P. flexilis. To design primers, we utilized sequences of the internal transcribed spacer (ITS) region and single-copy gene (RH_2175) of the TCP-1/cpn60 chaperonin family searched through genomes of related species. In addition to the DNA of target and non-target fungal species that were used for primer assays, environmental samples with next generation sequencing data were used to evaluate primer sensitivity. Direct amplification using ITS primer pairs generated 248–260 bp amplicons and successfully differentiated the needle pathogens used in this study. Nested amplification of single-copy gene RH_2175 primer pairs which produced 409–527 bp amplicons detected Rhytismataceae species and discriminated both Lophodermella pathogens on P. contorta var. latifolia, respectively. While ITS-based primers had higher sensitivity than the 2175-based primers, both primer sets for L. concolor and L. montivaga detected their respective pathogens in asymptomatic and symptomatic needles. These molecular tools can help monitor and assess needle diseases for forest management and phytosanitary regimes.

Comparative genomics of host-specialized populations of Corynespora cassiicola causing target spot epidemics in the southeastern United States.

Numerous plant-pathogenic fungi secrete necrotrophic effectors (syn. host-selective toxins) that are important determinants of pathogenicity and virulence in species that have a necrotrophic lifestyle. Corynespora cassiicola is a necrotrophic fungus causing emerging target spot epidemics in the southeastern United States (US). Previous studies revealed that populations of C. cassiicola from cotton, soybean, and tomato are clonal, host specialized and genetically distinct. Additionally, cassiicolin - the necrotrophic effector identified in some C. cassiicola isolates - is an important toxin for virulence on rubber. It is encoded by seven Cas gene variants. Our goal was to conduct comparative genomic analyses to identify variation among putative necrotrophic effector genes and to determine if lack of one of the mating-types explained clonal populations in C. cassiicola causing outbreaks in the southeastern US and the apparent absence of sexual reproduction worldwide. A total of 12 C. cassiicola genomes, with four each from isolates from tomato, soybean, and cotton, were sequenced using an Illumina Next Seq platform. Each genome was assembled de novo, compared with the reference genome from rubber, and searched for known Cas, and other gene clusters with homologs of secondary metabolites. Cas2 and/or Cas6 were present in isolates from soybean in the southeastern US, whereas Cas1 and Cas2 were present in isolates from cotton in the southeastern US. In addition, several toxin genes, including the T-toxin biosynthetic genes were present in all C. cassiicola from cotton, soybean, and tomato. The mating-type locus was identified in all of the sequenced genomes, with the MAT1-1 idiomorph present in all cotton isolates and the rubber isolate, whereas the MAT1-2 idiomorph was present in all soybean isolates. We developed a PCR-based marker for mating-type in C. cassiicola. Both mating types were present in isolates from tomato. Thus, C. cassiicola has both mating-types necessary for sexual reproduction, but the absence of both mating-types within soybean and cotton populations could explain clonality in these populations. Variation in necrotrophic effectors may underlie host specialization and disease emergence of target spot on cotton, soybean, and tomato in the southeastern US.

Establishment of a set of wheat-rye addition lines with resistance to stem rust.

Complete new wheat-rye disomic, telosomic addition lines and various chromosomal aberrations were developed and characterized by molecular cytogenetic method as novel chromosome engineering materials. A new stem rust resistance (Ug99) gene was located on 3RL. Wheat stem rust, caused by Puccinia graminis f. sp. tritici (Pgt), is a devastating fungal disease worldwide. A recently emerged great threat to global wheat production is Pgt strain Ug99 and its derivatives, which have overcome most of the commonly used resistance genes. Rye (Secale cereale L.), closely related to wheat (Triticum aestivum L.), is a significant and valuable resource of resistance genes for wheat germplasm improvement. It is of great importance and urgency to identify new resistance gene sources of rye and transfer them into wheat. In this study, two complete sets of wheat-rye addition lines were established through wide hybridization, chromosome doubling and backcrossing. A wheat-rye 3RL telosomic addition line was identified with high resistance to stem rust strain Ug99. PCR-based markers specific for the rye chromosome were developed. Furthermore, abundant chromosomal aberrations such as minichromosomes, ring chromosomes as well as centromere reduction and expansion were identified in the progeny of wheat-rye addition lines by multicolor GISH and FISH. The line carrying a novel resistance gene to stem rust can be utilized as a bridge material for wheat disease resistance breeding. The chromosomal and centromeric variation within the wheat-rye hybrids can further contribute to genetic diversity of their offspring.

Identification, High-Density Mapping, and Characterization of New Major Powdery Mildew Resistance Loci From the Emmer Wheat Landrace GZ1.

Powdery mildew is one of the most devastating diseases of wheat which significantly decreases yield and quality. Identification of new sources of resistance and their implementation in breeding programs is the most effective way of disease control. Two major powdery mildew resistance loci conferring resistance to all races in seedling and adult plant stages were identified in the emmer wheat landrace GZ1. Their positions, effects, and transferability were verified using two linkage maps (1,510 codominant SNP markers) constructed from two mapping populations (276 lines in total) based on the resistant GZ1 line. The dominant resistance locus QPm.GZ1-7A was located in a 90 cM interval of chromosome 7AL and explains up to 20% of the trait variation. The recessive locus QPm.GZ1-2A, which provides total resistance, explains up to 40% of the trait variation and was located in the distal part of chromosome 2AL. The locus was saturated with 14 PCR-based markers and delimited to a 0.99 cM region which corresponds to 4.3 Mb of the cv. Zavitan reference genome and comprises 55 predicted genes with no apparent candidate for the QPm.GZ1-2A resistance gene. No recessive resistance gene or allele was located at the locus before, suggesting the presence of a new powdery mildew resistance gene in the GZ1. The mapping data and markers could be used for the implementation of the locus in breeding. Moreover, they are an ideal base for cloning and study of host–pathogen interaction pathways determined by the resistance genes.

Validation of PCR-based Markers Associated with Sex Determination in Date Palm (Phoenix dactylifera L.)

Phoenix dactylifera (Date palm) is one of the essential commercial and economical fruit crops which mainly grows in hot arid areas. A good source of food, shelter, and energy, date palm is widely known due to its unique characteristics. The dioecious nature of date palm made it challenging to prognosticate its sex until they flower almost 5 to 8 years after plantation. Researchers have developed many strategies to determine sex, including morphological study, biochemical studies, molecular-based studies as sequence characterized amplified regions (SCAR), randomly amplified polymorphic DNA (RAPD) marker, and polymerase chain reaction (PCR) assays, but still more endeavours are integral. To foretell sex at the seedling stage, specific PCR primers were employed in this study. These primers were designed on sex-linked regions that determine their gender. Date palm DNA was extracted through the CTAB method. The DNA was then amplified through PCR using sex-linked specific primers. PCR products have run on gel electrophoresis using 1.2% agarose to score the bands for sex determination. Four primers were employed in this study to determine sex. Two primers were SCAR markers, and one was a sex-linked tetra primer. Tetra-primer amplified the double band fragment of 430 and 320bp respectively in male plants, while a band of 430bp was amplified in female plants. The other primer named ALAMER1 was amplified by the single band fragment of 186bp in males with no band in females. The fourth primer, named as SCARdp, amplified the band fragment of 354bp fragment in males. These SCAR markers were established to be male-specific. Sex-linked markers provide a platform for robust, efficient, and accurate determination of sex in date palms.