Search Results (17)

Germplasm improvement is essential for maize breeding. Currently, intra-heterotic-group crossing is the major method for germplasm improvement, while inter-heterotic-group crossing is also used in breeding but not in a systematic way. In this study, five inbred lines from four heterotic groups were used to develop a connected segregating population through inter-heterotic-group line crossing (CSPIC), which comprised 5 subpopulations with 535 doubled haploid (DH) lines and 15 related test-cross populations including 1568 hybrids. Significant genetic variation was observed in most subpopulations, with several DH populations exhibiting superior phenotypes regarding traits such as plant height (PH), ear height (EH), days to anthesis (DTA), and days to silking (DTS). Notably, 10.8% of hybrids in the population POP5/C229 surpassed the high-yielding hybrid ND678 (CK). To reduce field planting costs and quickly screen for the best inter-heterotic-group DH lines and test-cross hybrids, we assessed the accuracy of genomic selection (GS) for within- and between-population predictions in the DH populations and the test-cross populations. Within the DH or the hybrid population, the prediction accuracy varied across populations and traits, with an average hybrid yield prediction accuracy of 0.41, reaching 0.54 in POP5/Z58. In the cross DH population predictions, the prediction accuracy of the half-sib population exceeded that of the non-sib cross population prediction, with the highest accuracy observed when the non-shared parents were from the same heterotic group, and the average phenotypic prediction accuracies of POP3 predicting POP2 and POP2 predicting POP3 were 0.54 and 0.45, respectively. In the cross hybrid population predictions, the accuracy was highest when both the training and the test sets came from the same DH populations, with an average accuracy of 0.43. The proportion of shared polymorphisms with respect to SNPs between the training and the test sets (PSP) exhibited a significant and strong correlation with the prediction accuracy of cross population prediction. This study demonstrates the feasibility of creating new heterotic groups through inter-heterotic-group crossing in germplasm improvement, and some cross population prediction patterns exhibited excellent prediction accuracy. Full article

Haploid breeding can shorten the breeding period of new maize varieties and is an important means to increase maize yield. In the breeding program, a large number of haploid seeds need to be screened, and this step is mainly achieved manually, which hinders the industrialization of haploid maize breeding. This article aims to develop a multispectral camera to identify the haploid seeds automatically. The camera was manufactured by replacing narrow-band filters of the ordinary CCD camera, and the RGB, 405 nm, 980 nm and 1050 nm images of haploid or diploid seeds were simultaneously captured (the characteristic wavelengths were determined according to color and high-oil markers of maize). The performance was tested using four maize varieties with the two genetic markers. The results show that the developed multispectral camera significantly improved the recognition accuracy of haploid maize seeds to 92.33%, 97.33%, 97% and 93.33% for the TYD1903, TYD1904, TYD1907 and TYD1908 varieties, respectively. The cameras in the near-infrared region (wavelengths of 980 nm and 1050 nm) achieved better performance for the varieties of high-oil marker, with an increase of 0.84% and 1.5%, respectively. These results demonstrate the strong potential of the multispectral imaging technology in the haploid seed identification of maize. Full article

Candida albicans is a major human pathogenic fungus that is distinguished by its capability to switch from a yeast to a hyphal morphology under different conditions. Here, we analyze the cellular effects of high concentrations of the iron chelator bathophenanthroline disulfonate (BPS). BPS inhibits cellular growth by withholding iron, but when iron chelation is overcome by the addition of hemoglobin as an iron source, the cells resume growth as hyphae. The BPS hyphal induction pathway was characterized by identifying the hyphal-specific transcription factors that it requires and by a forward genetic screen for mutants that fail to form hyphae in BPS using a transposon library generated in a haploid strain. Among the mutants identified are the DYRK1-like kinase Yak1 and Orf19.384, a homolog of the DYRK1-associated protein WDR68/DCAF7. Orf19.384 nuclear localization depends on Yak1, similar to their mammalian counterparts. We identified the hyphal suppressor transcription factor Sfl1 as a candidate target of Yak1-Orf19.384 and show that Sfl1 modification is similarly affected in the yak1 and orf19.384 mutant strains. These results suggest that DYRK1/Yak1 and WDR68/Orf19.384 represent a conserved protein pair that regulates cell differentiation from fungi to animals. Full article

Somatic embryogenesis (SE) has many applications in grapevine biotechnology including micropropagation, eradicating viral infections from infected cultivars, mass production of hypocotyl explants for micrografting, as a continuous source for haploid and doubled haploid plants, and for germplasm conservation. It is so far the only pathway for the genetic modification of grapevines through transformation. The single-cell origin of somatic embryos makes them an ideal explant for mutation breeding as the resulting mutants will be chimera-free. In the present research, two combinations of plant growth regulators and different explants from flower buds at two stages of maturity were tested in regard to the efficiency of callusing and embryo formation from the callus produced in three white grape cultivars. Also, the treatment of somatic embryos with the chemical mutagen ethyl methanesulfonate (EMS) was optimised. Medium 2339 supplemented with β-naphthoxyacetic acid (5 μM) and 6-benzylaminopurine (BAP—9.0 μM) produced significantly more calluses than medium 2337 supplemented with 2,4-dichlorophenoxyacetic acid (4.5 µM) and BAP (8.9 µM) in all explants. The calluses produced on medium 2337 were harder and more granular and produced more SEs. Although the stage of the maturity of floral bud did not have a significant effect on the callusing of the explants, calluses produced from immature floral bud explants in the premeiotic stage produced significantly more SEs than those from more mature floral buds. Overall, immature ovaries and cut floral buds exposing the cut ends of filaments, style, etc., tested for the first time in grapevine SE, produced the highest percentage of embryogenic calluses. It is much more efficient to cut the floral bud and culture than previously reported explants such as anthers, ovaries, stigmas and styles during the short flowering period when the immature flower buds are available. When the somatic embryos of the three cultivars were incubated for one hour with 0.1% EMS, their germination was reduced by 50%; an ideal treatment considered to obtain a high frequency of mutations for screening. Our research findings will facilitate more efficient SE induction in grapevines and inducing mutations for improving individual traits without altering the genetic background of the cultivar. Full article

Rice plant height is an agricultural trait closely related to biomass, lodging tolerance, and yield. Identifying quantitative trait loci (QTL) regions related to plant height regulation and developing strategies to screen potential candidate genes can improve agricultural traits in rice. In this study, a double haploid population (CNDH), derived by crossing ‘Cheongcheong’ and ‘Nagdong’ individuals, was used, and a genetic map was constructed with 222 single-sequence repeat markers. In the RM3482-RM212 region on chromosome 1, qPh1, qPh1-1, qPh1-3, qPh1-5, and qPh1-6 were identified for five consecutive years. The phenotypic variance explained ranged from 9.3% to 13.1%, and the LOD score ranged between 3.6 and 17.6. OsPHq1, a candidate gene related to plant height regulation, was screened in RM3482-RM212. OsPHq1 is an ortholog of gibberellin 20 oxidase 2, and its haplotype was distinguished by nine SNPs. Plants were divided into two groups based on their height, and tall and short plants were distinguished and clustered according to the expression level of OsPHq1. QTLs and candidate genes related to plant height regulation, and thus, biomass regulation, were screened and identified in this study, but the molecular mechanism of the regulation remains poorly known. The information obtained in this study will help develop molecular markers for marker-assisted selection and breeding through rice plant height control. Full article

Genotyping by sequencing (GBS) was used to reveal the inherent genetic variation within the haploid fungi Sarocladium zeae isolated from diverse Zea germplasm, including modern Zea mays and its wild progenitors—the teosintes. In accordance with broad host relationship parameters, GBS analysis revealed significant host lineages of S. zeae genetic diversity, indicating that S. zeae genetic variation may associate with different evolutionary histories of host species or varieties. Based on a recently identified PKS-NRPS gene responsible for pyrrocidine biosynthesis in S. zeae fungi, a novel PCR assay was developed to discriminate pyrrocidine-producing S. zeae strains. This molecular method for screening bioactive strains of S. zeae is complementary to other approaches, such as chemical analyses. An eGFP-labelled S. zeae strain was also developed to investigate the endophytic transmission of S. zeae in Z. mays seedlings, which has further improved our understanding of the transmission modes of S. zeae endophytes in maize tissues. Full article

Photosynthesis is an important factor in determining the yield of rice. In particular, the size and efficiency of the photosynthetic system after the heading has a great impact on the yield. Research related to high-efficiency photosynthesis is essential to meet the growing demands of crops for the growing population. Chlorophyll is a key molecule in photosynthesis, a pigment that acts as an antenna to absorb light energy. Improvement of chlorophyll content characteristics has been emphasized in rice breeding for several decades. It is expected that an increase in chlorophyll content may increase photosynthetic efficiency, and understanding the genetic basis involved is important. In this study, we measured leaf color (CIELAB), chlorophyll content (SPAD), and chlorophyll fluorescence, and quantitative trait loci (QTL) mapping was performed using 120 Cheongcheong/Nagdong double haploid (CNDH) line after the heading date. A major QTL related to chlorophyll content was detected in the RM26981-RM287 region of chromosome 11. OsbHLHq11 was finally selected through screening of genes related to chlorophyll content in the RM26981-RM287 region. The relative expression level of the gene of OsbHLHq11 was highly expressed in cultivars with low chlorophyll content, and is expected to have a similar function to BHLH62 of the Gramineae genus. OsbHLHq11 is expected to increase photosynthetic efficiency by being involved in the chlorophyll content, and is expected to be utilized as a new genetic resource for breeding high-yield rice. Full article

In vitro plant tissue culture and biotechnology used to assist and support the development of plant breeding when classical methods of propagation must be accelerated or it was necessary to overcome barriers inaccessible by classical approaches. In asparagus, to improve multiple breeding tasks, a high number of in vitro methods have been used, such as plant regeneration methods through organogenesis, embryogenesis, manipulation of ploidy, protoplast isolation, genetic manipulation (protoplast fusion, genetic transformation), embryo rescue and germplasm preservation (in vitro, in vitro slow growth, cryopreservation). Plant tissue culture methods can overcome multiple problems in asparagus breeding such as, barriers of self and cross-incompatibility between asparagus species through embryo rescue of interspecific hybrids and protoplast fusion or genetic transformation, introgression of new genes, clonal propagation of elite genotypes of asparagus, mass screening, and the generation of haploid and polyploid genotypes, among others, becoming the tool of choice for asparagus breeding programs. Some of these in vitro methods are still under development. Full article

As a wild ancestor of cultivated rice, Oryza rufipogon is domesticated into cultivated rice Oryza sativa, many agricultural traits are newly created or disappear. In particular, in wild rice, awn protects from predators and is easily blown by the wind and used as a means of propagation. However, awns gradually disappeared as they were breeding from wild rice to cultivated rice. Since awn development is disadvantageous to rice yield, it is important to understand the genetic basis related to awn development. In addition, characterization of the genes associated with awn development is helpful in analyzing the genetic relationships of rice from ancient times to the present for the regulatory mechanisms of awn formation. QTL analysis identified RM14330-RM218 on chromosome 3 using a 120 Cheongcheong/Nagdong double haploid population. Through screening of genes related to awn development in RM-14330-RM218, it is indicated that OsDRPq3 is a causal gene that can be involved in awn development. OsDRPq3 transcription level is maintained high in long awn and less yield populations during the panicle formation stage, the period during awn development. Moreover, the sequence of OsDRPq3 has high homology with the drooping protein leaf. This study provides a new resource for phylogenetic research of rice and exploration of awn development. Full article

Rice tillers are one of the most important traits for the yield and development of rice, although little is known about its mode of inheritance. Tiller numbers were recorded every 7 days a total of nine times, starting 30 days after transplantation. Quantitative trait locus (QTL) based analysis on a set of double haploid population derivatives of a cross between the Cheongcheong and Nagdong varieties identified a major effect of locus RM18130–RM3381 on chromosome 5, which was expressed in eight different growth stages. Within the target region RM18130–RM3381 (physical distance: 2.08 Mb), 61 candidate genes were screened by annotation. Among the candidate genes, Os05g0230700 (named OsIAA17q5), which belongs to the family of auxin-responsive genes, was selected as a target. Auxin promotes cell division and meristem maintenance and is an effective plant regulator which influences plant growth and development by altering the expression of various genes. OsIAA17q5 is expected to control the number of tillers. The present study provides further understanding of the basic genetic mechanisms that selectively express the control of tiller numbers in different growth stages, as well as provides valuable information for future research aimed at cloning the target gene. These results may contribute to developing a comprehensive understanding of the basic genetic processes regulating the developmental behavior of tiller numbers in rice. Full article

The domestic silkworm Bombyx mori is extensively studied as a model organism for lepidopteran genetics and has an economic value in silk production. Silkworms also have applications in biomedical and cosmetic industries, and the production of mutant B. mori strains significantly enhances basic and applied silkworm research. In recent years, CRISPR/Cas9 technology is being rapidly adopted as the most efficient molecular tool for generating silkworm lines carrying mutations in target genes. Here we illustrate a complete and efficient workflow to screen, characterize rapidly and follow mutations through generations, allowing the generation of B. mori lines, stably inheriting single CRISPR/Cas9-induced mutations. This approach relies on the use of different molecular methods, the heteroduplex assay, cloning followed by Sanger sequencing, and the amplification refractory mutation system PCR. The use of these methodologies in a sequential combination allows the identification of CRISPR/Cas9-induced mutations in genes mapping on both autosomes and sex chromosomes, and the selection of appropriate individuals to found stable mutant B. mori lines. This protocol could be further applied to screen CRISPR/Cas9 mutations in haploid insects. Full article

Although orthopoxviruses (OPXV) are known to encode a majority of the genes required for replication in host cells, genome-wide genetic screens have revealed that several host pathways are indispensable for OPXV infection. Through a haploid genetic screen, we previously identified several host genes required for monkeypox virus (MPXV) infection, including the individual genes that form the conserved oligomeric Golgi (COG) complex. The COG complex is an eight-protein (COG1–COG8) vesicle tethering complex important for regulating membrane trafficking, glycosylation enzymes, and maintaining Golgi structure. In this study, we investigated the role of the COG complex in OPXV infection using cell lines with individual COG gene knockout (KO) mutations. COG KO cells infected with MPXV and vaccinia virus (VACV) produced small plaques and a lower virus yield compared to wild type (WT) cells. In cells where the KO phenotype was reversed using a rescue plasmid, the size of virus plaques increased demonstrating a direct link between the decrease in viral spread and the KO of COG genes. KO cells infected with VACV displayed lower levels of viral fusion and entry compared to WT suggesting that the COG complex is important for early events in OPXV infection. Additionally, fewer actin tails were observed in VACV-infected KO cells compared to WT. Since COG complex proteins are required for cellular trafficking of glycosylated membrane proteins, the disruption of this process due to lack of individual COG complex proteins may potentially impair the virus-cell interactions required for viral entry and egress. These data validate that the COG complex previously identified in our genetic screens plays a role in OPXV infection. Full article

Ebola Virus Disease (EVD) is one of the most lethal transmissible infections, characterized by a high fatality rate, and caused by a member of the Filoviridae family. The recent large outbreak of EVD in Western Africa (2013–2016) highlighted the worldwide threat represented by the disease and its impact on global public health and the economy. The development of highly needed anti-Ebola virus antivirals has been so far hampered by the shortage of tools to study their life cycle in vitro, allowing to screen for potential active compounds outside a biosafety level-4 (BSL-4) containment. Importantly, the development of surrogate models to study Ebola virus entry in a BSL-2 setting, such as viral pseudotypes and Ebola virus-like particles, tremendously boosted both our knowledge of the viral life cycle and the identification of promising antiviral compounds interfering with viral entry. In this context, the combination of such surrogate systems with large-scale small molecule compounds and haploid genetic screenings, as well as rational drug design and drug repurposing approaches will prove priceless in our quest for the development of a treatment for EVD. Full article

The identification of host cellular genes that act as either proviral or antiviral factors has been aided by the development of an increasingly large number of high-throughput screening approaches. Here, we review recent advances in which these new technologies have been used to interrogate host genes for the ability to impact bunyavirus infection, both in terms of technical advances as well as a summary of biological insights gained from these studies. Full article

Using biological fertilizers and pesticides based on beneficial soil microbes in order to reduce mineral fertilizers and chemical pesticides in conventional agriculture is still a matter of debate. In this regard, a European research project seeks to elucidate the role of root-endophytic fungi and to develop molecular tools to trace and quantify these fungi in the rhizosphere and root tissue. To do this, the draft genome sequence of the biocontrol fungus Trichoderma virens (T. virens) was screened for simple sequence repeats (SSRs) and primers were developed for 12 distinct loci. Primers were evaluated using a global collection of ten isolates where an average of 7.42 alleles per locus was detected. Nei’s standard genetic distance ranged from 0.18 to 0.27 among the isolates, and the grand mean of haploid diversity in AMOVA analysis was 0.693 ± 0.019. Roots of tomato plants were inoculated with different strains and harvested six weeks later. Subsequent PCR amplification identified root-endophytic strains and co-colonization of roots by different strains. Markers were applied to qPCR to quantify T. virens strains in root tissue and to determine their identity using allele-specific melting curve analysis. Thus, the root-endophytic lifestyle of T. virens was confirmed, strains in roots were quantified and simultaneous colonization of roots by different strains was observed. Full article