Utilization of Molecular Markers for Rice Breeding

Rice is the staple food for more than half of the world's population. Rice production in 2050 must increase by at least 50% to keep up with the population growth. Efforts to increase rice production continue using various strategies. Breeders apply multiple approaches including application of molecular markers in developing varieties better than the previous ones. Since the discovery of the restriction fragment length polymorphism (RFLP) markers in 1980s and the development of polymerase chain reaction (PCR) method, many types of molecular markers have been developed and applied to various crops including rice. Various molecular approaches to map genetic loci associated with rice superior traits were conducted. The mapped loci are very useful for rice breeding purposes. This paper reports the results of mapping and breeding economically important traits in rice, mainly those related to abiotic stresses, agronomic traits, yield, and yield quality. These included characters of semidwarf stature, aromatic grain, high yield potential, eating quality, higher Zn and Fe grain, more tolerant to abiotic stresses, such as salinity, drought, phosphate deficiency, Al toxicity and Fe toxicity, submergence, as well as early maturity character. The mapped characters can be transferred using marker-assisted backcrossing (MABC) method into cultivated rice genotypes well-adopted by farmers. Several countries including Indonesia have benefited from this breeding method, and Indonesia have released several rice varieties developed through MABC. These include rice varieties such as Code, Angke, Inpari 30, Inpari Blas, Inpari HDB, Bio Patenggang Agritan, and Bioni 63 Ciherang Agritan

A systematic review of Toxoplasma gondii genotypes in Gallus gallus domesticus worldwide: The focus is Brazil

Toxoplasma gondii was initially classified in three main lineages related to its virulence: Types I, II and III. The recombination of genes during sexual cycle in felids gut led to more than 200 genotypes, found in ToxoDB database, using 11 RFLP markers. Free-range chickens are good bioindicators of soil contamination with T. gondii oocysts. In this sense, there are systematic reviews regarding data of genetic characterization of this parasite in felines and ruminants, but not in chickens heretofore, what makes this work necessary. A systematic review in the literature was performed with papers published prior to September 21st, 2020. The main inclusion criteria was the presence of T. gondii genotypes, isolated strictly from free-range chickens, in experimental works. Initially, a total of 1,343 studies related to the terms were identified on databases and 30 studies were selected to be systematically reviewed. A total of 561 isolates of T. gondii from 6,356 free-range chickens were analyzed for genotyping, revealing 190 genotypes. ToxoDB #59 and #2 were the most frequent in America, #1 was the most frequent in Africa and 3 atypical isolates from genotype ToxoDB #9 were found in Asia. There is not data from Europe and Oceania. The majority of studies were Brazilian (16/30). A total of 68 RFLP genotypes were recognized among the 561 isolates’ DNAs analyzed from the 30 studies. Some studies show new genotypes never described before, which reinforces the idea that some years from now, even more new genotypes will be isolated, due to progressive genetic recombination. The large amount of undefined genotypes makes it necessary to perform Nested PCR technique when genotyping. Moreover, the lack of data in Continents such as Europe, Asia and Oceania makes it necessary to perform new isolating and genotyping studies in these places.

Molecular Species Identification of Six Forensically Important Iranian Flesh Flies (Diptera)

Background: Flesh flies (Diptera: Sarcophagidae) are considered as myiasis agents and important evidences in forensic investigations. However, their use has been restricted because, at all larval stages and female adults, morphological species identification is difficult or very challenging. This study investigated to test utility of mitochondrial cytochrome oxidase subunit I (mt-COI) sequences for differentiation of six forensically important Iranian flesh flies namely, Sarcophaga crassipalpis, S. flagellifera, S. hirtipes, S. aegyptica, S. africa and S. argyrostoma. Methods: Male specimens were morphologically identified to species level and then the genomic DNA of the flies were extracted and subjected to polymerase chain reaction (PCR) against mt-COI gene. The PCR products were sequenced and the obtained sequences were analyzed for the species specific restriction fragment length polymorphisms (RFLPs). Results: Rate of genetic variation between species was 6–10% which was enough to find restriction enzymes (RE) that were able to produce species-specific RFLP profiles. Combinations of three REs: BsrFI, RsaI and HinfI, provided diagnostic bands for identification of the six Sarcophaga species. Conclusions: The results of this study showed that molecular markers such as RFLPs enhancing the use of evidence from flesh flies in forensic investigation. However, lack proper restriction sites in the COI region inhibited introduction of a single restriction enzyme for easy species identification. It is recommended to apply larger part of DNA such as combination of COI and COII genes to provide better RFLP markers for species identification of flesh flies.

Link between PON 1 gene mutation and recurrent miscarriage among women exposed to pesticides in North India is insignificant

Recurrent miscarriage is a loss of disconcerting disorder characterized by RPL (recurrent pregnancy loss) of pregnancy, affecting around 1-2% of couples trying to conceive. Exposure to pesticide affects spontaneous abortion, and infertility in women. Placental oxidative stress is often linked to miscarriage. Therefore, it is of interest to link PON1 (Q1922R) polymorphism with recurrent pregnancy loss. We selected 200 subjects in which 100 patients with RPL having consecutive 2 or more miscarriages and 100 healthy controls from the northern India for this study. Blood samples were collected for DNA isolation and further assessment. Genotyping of the Q1922R polymorphism was completed using the RFLP markers. The digested PCR product size was 99 bp (control). The heterozygous fragments were found to be 66 and 33 bp homozygous mutants. It was observed that allele frequency homozygous (TT) was more prevalent among control than the case groups among the healthy north-Indian population. However, heterozygous group (Tt) was more in cases compared to control groups as well as homozygous mutant was observed high in control in than case (CI-0.3 to 1.3).

Comparative Molecular Description of a Novel GST Gene in Culex pipiens (Diptera: Culicidae)

Repeated exposure to insecticides, particularly pyrethroids and organophosphates, has resulted in the development of insecticide resistance in the mosquito Culex pipiens, a primary disease vector. Glutathione S-transferase (GST) is involved in the phase II detoxification of numerous xenobiotics, including insecticides. In this study, a GST gene (CPIJ002678) was amplified, sequenced, and used in comprehensive molecular analyses ending up in development of a rapid assay to distinguish more tolerant individuals from susceptible Culex pipiens using the Restriction Fragment Length Polymorphism (RFLP) technique. Field collected Culex pipiens strains from untreated areas, organophosphates-treated areas and a lab strain reared for many generations, all were used in CDC bottle bioassays to evaluate the susceptibility status of the studied individuals to malathion insecticide. Interestingly, both field sites collected groups showed high levels of resistance at the malathion diagnostic time. Gene amplification, and bidirectional direct sequencing results were analyzed. Compared with the reference genome sequence, the pairwise alignment of the amplified sequences showed 96.6% similarity to the reference sequence in the GenBank database. The confirmed gene sequences were assembled and aligned using various bioinformatic softwares. The assembled contigs were used in NEBcutter V2.0 for constructing restriction maps and checked for the availability of differences (if present) between susceptible and more tolerant strains. Specific molecular RFLP markers were successfully recognized to differentiate the more tolerant from the susceptible Culex pipiens phenotypes.

Identification and Validation of Candidate Genes Conferring Resistance to Downy Mildew in Maize (Zea mays L.)

Downy mildew (DM) is a major disease of maize that causes significant yield loss in subtropical and tropical regions around the world. A variety of DM strains have been reported, and the resistance to them is polygenically controlled. In this study, we analyzed the quantitative trait loci (QTLs) involved in resistance to Peronosclerospora sorghi (sorghum DM), P. maydis (Java DM), and Sclerophthora macrospora (crazy top DM) using a recombinant inbred line (RIL) from a cross between B73 (susceptible) and Ki11 (resistant), and the candidate genes for P. sorghi, P. maydis, and S. macrospora resistance were discovered. The linkage map was constructed with 234 simple sequence repeat (SSR) and restriction fragment length polymorphism (RFLP) markers, which was identified seven QTLs (chromosomes 2, 3, 6, and 9) for three DM strains. The major QTL, located on chromosome 2, consists of 12.95% of phenotypic variation explained (PVE) and a logarithm of odds (LOD) score of 14.12. Sixty-two candidate genes for P. sorghi, P. maydis, and S. macrospora resistance were obtained between the flanked markers in the QTL regions. The relative expression level of candidate genes was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) using resistant (CML228, Ki3, and Ki11) and susceptible (B73 and CML270) genotypes. For the 62 candidate genes, 15 genes were upregulated in resistant genotypes. Among these, three (GRMZM2G028643, GRMZM2G128315, and GRMZM2G330907) and AC210003.2_FG004 were annotated as leucine-rich repeat (LRR) and peroxidase (POX) genes, respectively. These candidate genes in the QTL regions provide valuable information for further studies related to P. sorghi, P. maydis, and S. macrospora resistance.

Prevalence and genotyping of Toxoplasma gondii in stray cats in Mashhad area, Iran

Background Cats as a definitive host have an important role in the epidemiology of toxoplasmosis in humans and animals. The aim of the study was to determine the frequency of Toxoplasma gondii infection and isolate and identify the genotypes of T. gondii in stray cats in the Mashhad suburb. Methods From April 2016 to August 2017, 175 fecal samples from stray cats and 31 brain samples from cats killed in driving accidents were collected. The fecal samples were examined by fecal flotation technique and T. gondii-specific PCR. The brain samples were investigated by T. gondii-specific PCR and consequently examined by mice bioassay. The DNA of T. gondii isolated was genotyped using SAG1, SAG2, SAG3, BTUB and GRA6 as PCR-restriction fragment length polymorphism (PCR-RFLP) markers. Results In the present study, Toxoplasma-like oocysts were microscopically observed in 2.2% (4/175) fecal samples. The presence of Toxoplasma oocysts was confirmed in one microscopy-positive sample by PCR. In addition, T. gondii DNA was detected in 4% (7/175) microscopy-negative samples using PCR. T. gondii was isolated from one brain PCR-positive sample by mice bioassay. The isolate was avirulent and many T. gondii cysts were observed in mice brain. The isolate was successfully genotyped by PCR-RLFP analysis. The isolated genotyped was type II. Besides, eight Toxoplasma-positive fecal samples contained insufficient DNA and only amplified at SAG-3 locus in PCR. These samples were also showed type II pattern at this locus. Conclusions Parasitological and molecular results showed low frequency of Toxoplasma infection in the stray cats, and identified the genotype of T. gondii isolate as type II, for the first time in Mashhad area, Khorasan Razavi Province.

Comparison between single and multi-locus approaches for specimen identification in Mytilus mussels

Mytilus mussels have been the object of much research given their sentinel role in coastal ecosystems and significant value as an aquaculture resource appreciated for both, its flavour and nutritional content. Some of the most-studied Mytilus species are M. edulis, M. galloprovincialis, M. chilensis and M. trossulus. As species identification based on morphological characteristics of Mytilus specimens is difficult, molecular markers are often used. Single-locus markers can give conflicting results when used independently; not all markers differentiate among all species, and the markers target genomic regions with different evolutionary histories. We evaluated the concordance between the PCR-RFLP markers most commonly-used for species identification in mussels within the Mytilus genus (Me15-16, ITS, mac-1, 16S rRNA and COI) when used alone (mono-locus approach) or together (multi-locus approach). In this study, multi-locus strategy outperformed the mono-locus methods, clearly identifying all four species and also showed similar specimen identification performance than a 49 SNPs panel. We hope that these findings will contribute to a better understanding of DNA marker-based analysis of Mytilus taxa. These results support the use of a multi-locus approach when studying this important marine resource, including research on food quality and safety, sustainable production and conservation.

Prevalence and genotyping of Toxoplasma gondii in stray cats in Mashhad area, Iran.

Background: Cats as a definitive host have an important role in the epidemiology of toxoplasmosis in humans and animals. The aim of the study was to determine the frequency of Toxoplasma gondii infection and isolate and identify the genotypes of T. gondii in stray cats in the Mashhad suburb. Methods: From April 2016 to August 2017, 175 fecal samples from stray cats and 31 brain samples from cats killed in driving accidents were collected. The fecal samples were examined by fecal flotation technique and T. gondii-specific PCR. The brain samples were investigated by T. gondii-specific PCR and consequently examined by mice bioassay. The DNA of T. gondii isolated was genotyped using SAG1, SAG2, SAG3, BTUB and GRA6 as PCR-restriction fragment length polymorphism (PCR-RFLP) markers. Results: In the present study, Toxoplasma-like oocysts were microscopically observed in 2.2% (4/175) fecal samples. The presence of Toxoplasma oocysts was confirmed in one microscopy-positive sample by PCR. In addition, T. gondii DNA was detected in 4% (7/175) microscopy-negative samples using PCR. T. gondii was isolated from one brain PCR-positive sample by mice bioassay. The isolate was avirulent and many T. gondii cysts were observed in mice brain. The isolate was successfully genotyped by PCR-RLFP analysis .The isolated genotyped was type II. Besides, eight Toxoplasma-positive fecal samples contained insufficient DNA and only amplified at SAG-3 locus in PCR. These samples were also showed type II pattern at this locus. Conclusions: Parasitological and molecular results showed low frequency of Toxoplasma infection in the stray cats, and identified the genotype of T. gondii isolate as type II, for the first time in Mashhad area, Khorasan Razavi Province.

Prevalence and genotyping of Toxoplasma gondii in stray cats in Mashhad area, Iran.

Background: Cats as a definitive host have an important role in the epidemiology of toxoplasmosis in humans and animals. The aim of the study was to determine the frequency of Toxoplasma gondii infection and isolate and identify the genotypes of T. gondii in stray cats in Mashhad suburb. Methods: From April 2016 to August 2017, 175 fecal samples from stray cats and 31 brain samples from cats killed in driving accidents were collected. The fecal samples were examined by fecal flotation technique and T. gondii -specific PCR. The brain samples were investigated by T. gondii -specific PCR and consequently examined by mice bioassay. The DNA of T. gondii isolated was genotyped using SAG1, SAG2, SAG3, BTUB and GRA6 as PCR-restriction fragment length polymorphism (PCR-RFLP) markers. Results: In the present study, Toxoplasma -like oocysts were microscopically observed in 2.2% (4/175) fecal samples. The presence of Toxoplasma oocysts was confirmed in one microscopy-positive sample by PCR. In addition, T . gondii DNA was detected in 4% (7/175) microscopy-negative samples using PCR. T. gondii was isolated from one brain PCR-positive sample by mice bioassay. The isolate was avirulent and many T. gondii cysts were observed in mice brain. The isolate was successfully genotyped by PCR-RLFP analysis .The isolated genotyped was type II. Beside, eight Toxoplasma- positive fecal samples contained insufficient DNA and only amplified at SAG-3 locus in PCR. These samples were also showed type II pattern at this locus. Conclusions: Parasitological and molecular results showed low frequency of Toxoplasma infection in the stray cats, and identified the genotype of T. gondii isolate as type II, for the first time in Mashhad area, Khorasan Razavi Province.