Prospects for Knockout of MYB60, a Transcriptional Repressor of Anthocyanin Biosynthesis, in Brassicaceae Plants by Genome Editing

The Brassicaceae plant family contains many economically important crops such as Brassica napus L., Brassica rapa L., Brassica oleracea L., Brassica juncea L., Eruca sativa Mill., Camelina sativa L. and Raphanus sativus L. Insufficient data on the genetic regulation of agronomic traits in these species complicates the editing of their genomes. In recent years, the attention of the academic community has been drawn to anthocyanin hyperaccumulation. This trait is not only beneficial for human health, but can also increase plant resistance to stress. MYB transcription factors are the main regulators of flavonoid biosynthesis in plants. Some of them are well studied in Arabidopsis thaliana. The AtMYB60 gene is a transcriptional repressor of anthocyanin biosynthesis, and it also negatively impacts plant responses to drought stress. Myb60 is one of the least studied transcription factors with similar functions in Brassicaceae. There is a high degree of homology between predicted MYB60 genes of A. thaliana and related plant species. However, functions of these homologous genes have never been studied. Gene knockout by CRISPR/Cas technology remains the easiest way to perform genome editing in order to discover the role of individual plant genes. Disruption of genes acting as negative regulators of anthocyanin biosynthesis could result in color staining of plant tissues and an increase in stress tolerance. In the present study, we investigated the AtMYB60 gene and its homologs in Brassicaceae plants and suggested universal gRNAs to knockout these genes. Keywords: CRISPR, Brassicaceae, MYB60, knockout, anthocyanin

Whole-Genome Sequencing of 117 Chromosome Segment Substitution Lines for Genetic Analyses of Complex Traits in Rice

Rice is one of the most important food crops in Asia. Genetic analyses of complex traits and molecular breeding studies in rice greatly rely on the construction of various genetic populations. Chromosome segment substitution lines (CSSLs) serve as a powerful genetic population for quantitative trait locus (QTL) mapping in rice. Moreover, CSSLs containing target genomic regions can be used as improved varieties in rice breeding. In this study, we developed a set of CSSLs consisting of 117 lines derived from the recipient ‘Huanghuazhan’ (HHZ) and the donor ‘Basmati Surkb 89–15’ (BAS). The 117 lines were extensively genotyped by whole-genome resequencing, and a high-density genotype map was constructed for the CSSL population. The 117 CSSLs covered 99.78% of the BAS genome. Each line contained a single segment, and the average segment length was 6.02 Mb. Using the CSSL population, we investigated three agronomic traits in Shanghai and Hangzhou, China, and a total of 25 QTLs were detected in both environments. Among those QTLs, we found that RFT1 was the causal gene for heading date variance between HHZ and BAS. RFT1 from BAS was found to contain a loss-of-function allele based on yeast two-hybrid assay, and its causal variation was a P to S change in the 94th amino acid of the RFT1 protein. The combination of high-throughput genotyping and marker-assisted selection (MAS) is a highly efficient way to construct CSSLs in rice, and extensively genotyped CSSLs will be a powerful tool for the genetic mapping of agronomic traits and molecular breeding for target QTLs/genes.

Wheat Microbiome: Structure, Dynamics, and Role in Improving Performance Under Stress Environments

Wheat is an important cereal crop species consumed globally. The growing global population demands a rapid and sustainable growth of agricultural systems. The development of genetically efficient wheat varieties has solved the global demand for wheat to a greater extent. The use of chemical substances for pathogen control and chemical fertilizers for enhanced agronomic traits also proved advantageous but at the cost of environmental health. An efficient alternative environment-friendly strategy would be the use of beneficial microorganisms growing on plants, which have the potential of controlling plant pathogens as well as enhancing the host plant’s water and mineral availability and absorption along with conferring tolerance to different stresses. Therefore, a thorough understanding of plant-microbe interaction, identification of beneficial microbes and their roles, and finally harnessing their beneficial functions to enhance sustainable agriculture without altering the environmental quality is appealing. The wheat microbiome shows prominent variations with the developmental stage, tissue type, environmental conditions, genotype, and age of the plant. A diverse array of bacterial and fungal classes, genera, and species was found to be associated with stems, leaves, roots, seeds, spikes, and rhizospheres, etc., which play a beneficial role in wheat. Harnessing the beneficial aspect of these microbes is a promising method for enhancing the performance of wheat under different environmental stresses. This review focuses on the microbiomes associated with wheat, their spatio-temporal dynamics, and their involvement in mitigating biotic and abiotic stresses.

Susceptibility of almond (Prunus dulcis) cultivars to twig canker and shoot blight caused by Diaporthe amygdali

Twenty-five almond cultivars were assessed for susceptibility to Diaporthe amygdali, causal agent of twig canker and shoot blight disease. In laboratory experiments, growing twigs were inoculated with four D. amygdali isolates. Moreover, growing shoots of almond cultivars grafted onto INRA ‘GF-677’ rootstock were used in four-year field inoculations with one D. amygdali isolate. In both type of experiments, inoculum consisted of agar plugs with mycelium, which were inserted underneath the bark and the lesion lengths caused by the fungus were measured. Necrotic lesions were observed in the inoculated almond cultivars both in laboratory and field tests, confirming the susceptibility of all the evaluated cultivars to all the inoculated isolates of D. amygdali. Cultivars were grouped as susceptible or very susceptible according to a cluster analysis. The relationship between some agronomic traits and cultivar susceptibility was also investigated. Blooming and ripening times were found relevant variables to explain cultivars performance related to D. amygdali susceptibility. Late and very late blooming, and early and medium ripening cultivars were highly susceptible to D. amygdali. Our results may provide valuable information that could assist in ongoing breeding programs of this crop and additionally in the selection of cultivars for new almond plantations.

Improvement of Cercospora leaf spot and powdery mildew resistance of mungbean variety KING through marker-assisted selection

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.

The PCY-SAG14 phytocyanin module regulated by PIFs and miR408 promotes dark-induced leaf senescence in Arabidopsis

Leaf senescence is a critical process in plants and has a direct impact on many important agronomic traits. Despite decades of research on senescence-altered mutants via forward genetics and functional assessment of senescence-associated genes (SAGs) via reverse genetics, the senescence signal and the molecular mechanism that perceives and transduces the signal remain elusive. Here, using dark-induced senescence (DIS) of Arabidopsis leaf as the experimental system, we show that exogenous copper induces the senescence syndrome and transcriptomic changes in light-grown plants parallel to those in DIS. By profiling the transcriptomes and tracking the subcellular copper distribution, we found that reciprocal regulation of plastocyanin, the thylakoid lumen mobile electron carrier in the Z scheme of photosynthetic electron transport, and SAG14 and plantacyanin (PCY), a pair of interacting small blue copper proteins located on the endomembrane, is a common thread in different leaf senescence scenarios, including DIS. Genetic and molecular experiments confirmed that the PCY-SAG14 module is necessary and sufficient for promoting DIS. We also found that the PCY-SAG14 module is repressed by a conserved microRNA, miR408, which in turn is repressed by phytochrome interacting factor 3/4/5 (PIF3/4/5), the key trio of transcription factors promoting DIS. Together, these findings indicate that intracellular copper redistribution mediated by PCY-SAG14 has a regulatory role in DIS. Further deciphering the copper homeostasis mechanism and its interaction with other senescence-regulating pathways should provide insights into our understanding of the fundamental question of how plants age.

Genetic Variability and G×E Interactions in a Diverse Set of Groundnut Accessions

Background: Knowledge of the genetic diversity for various agronomic traits and their interaction with the environment and subsequent classification of genotypes will be beneficial for identification of divergent and stable sources of agronomic traits. Methods: A set of 96 groundnut germplasm accessions belonging to four botanical groups were evaluated for three years (2017 to 2019) for pod yield and component traits using AMMI analysis and subsequently accessions were classified based Euclidean cluster analysis. Result: Among different botanical groups, Virginia genotypes matured late and possessed high SPAD chlorophyll meter readings (SCMR) and pod yield compared to Spanish types. The component traits of pod maturity like days to flowering (first and 50%) showed low heritability and high genotype × environment interaction (GEI) and significant negatively affected sound mature kernel (SMK) and shelling per centage (SP). The cumulative contribution of environment and GEI component to the total variance was the highest in the expression of SP (67%) followed by days to maturity (54%) and days to 50% flowering (52%). Euclidean distance-based cluster analysis grouped the 96 accessions into five major clusters. Cluster I had accessions with higher pod yield, whereas cluster V contained accessions with low SLA, high SCMR and moderate pod yield. High yielding as well as stable accessions identified based on AMMI stability value (ASV) are NRCG 17332, 10076, 17268, 17197, 17108, 10106, 10089 and 17165. Trait specific as well as stable accessions identified in the present study can be useful donors for groundnut breeding programme.

Genetic Diversity and Population Structure Analysis in Early Generations Maize Inbreds Derived From Local Germplasm of Eastern Himalayan Regions Using Microsatellite Markers

North- Eastern parts of India fall under the Eastern Himalayan region and it is a diversity hotspot of many crops, including maize. Maize is an important traditional cereal crop grown in hill ecology of the region mainly for food, fodder and feed. To tap the potentiality of maize genetic resources in crop improvement programmes, assessment of genetic diversity is a basic requirement. Hence, in the present study, assessment of genetic diversity in thirty early generation maize inbreds developed from different germplasm of NE India was taken up using genome wide distributed fifty two microsatellite markers. The marker analysis revealed a large variation with a total of 189 alleles with an average of 3.63 alleles per marker locus. The allele size ranged from 50 bp ( phi 036 ) to 295 bp ( p 101049 ) which revealed a high level of genetic diversity among the loci. The PIC value ranged from 0.17 ( umc 1622 ) to 0.76 ( umc 1153 ) with an average value of 0.49. The value of expected Heterozygosity (H Exp ) ranged from 0.19 to 0.80 with an average of 0.57, whereas the Observed Heterozygosity (H Obs ) ranged from 0 to 0.89 with a mean of 0.14.The genetic dissimilarity between the genotype pairs ranged from 0.40 to 0.64 with a mean value of 0.57. Cluster analysis resolved the inbreds into three distinct sub-clusters. Similarly, population structure analysis also classified the inbred lines into three-subpopulations. Marker-trait associations showed a total of twelve SSR markers significantly associated with seven agronomic traits. From the present study, wide genetic variability was found among the maize inbreds with high potential to contribute new beneficial and unique alleles in genetic enhancement program of maize in India and particularly, in NE region.

Identification of Genetic Loci on Chromosome 4B for Improving the Grain Number per Spike in Pre-Breeding Lines of Wheat

The grain number per spike (GNPS) is an important yield component, and much attention is given to the increase in GNPS for current yield improvement of common wheat. Here, a panel of 259 pre-breeding lines and elite commercial varieties were collected for the investigation of 12 agronomic traits, especially for spike-related traits, with 2-year replicates. The high correlation between GNPS and kernel number per spikelet (KNS) suggested that the high GNPS trait in our pre-breeding lines was mainly controlled by grain set number per spikelet. Genome-wide association studies (GWAS) using the 660K SNP genotyping assay suggested that a major locus on chromosomes 4BS contributed to the high GNPS trait, which contributed to 33% and 48% of the variation in KNS and GNPS, respectively. A good diagnostic KASP marker AX-109286577 flanking the 4BS locus was developed for easy selection of the large spike trait. Taken together, the results suggested that untapped rare allele variation in our pre-breeding lines can be used for improvement of the yield component of set grain number per spike.

Variation of Macro- and Microelements, and Trace Metals in Spring Wheat Genetic Resources in Siberia

Western Siberia is one of the major spring wheat regions of Russia, cultivating over 7 Mha. The objective of the study was to evaluate the variation of macro- and microelements, and of trace metals in four distinct groups of genetic resources: primary synthetics from CIMMYT (37 entries), primary synthetics from Japan (8), US hard red spring wheat cultivars (14), and material from the Kazakhstan–Siberian Network on Spring Wheat Improvement (KASIB) (74). The experiment was conducted at Omsk State Agrarian University, using a random complete block design with four replicates in 2017 and 2018. Concentrations of 15 elements were included in the analysis: macroelements, Ca, K, Mg, P, and S; microelements, Fe, Cu, Mn, and Zn; toxic trace elements, Cd, Co, Ni; and trace elements, Mo, Rb, and Sr. Protein content was found to be positively correlated with the concentrations of 11 of the elements in one or both years. Multiple regression was used to adjust the concentration of each element, based on significant correlations with agronomic traits and macroelements. All 15 elements were evaluated for their suitability for genetic enhancement, considering phenotypic variation, their share of the genetic component in this variation, as well as the dependence of the element concentration on other traits. Three trace elements (Sr, Mo, and Co) were identified as traits that were relatively easy to enhance through breeding. These were followed by Ca, Cd, Rb, and K. The important biofortification elements Mn and Zn were among the traits that were difficult to enhance genetically. The CIMMYT and Japanese synthetics had significantly higher concentrations of K and Sr, compared to the local check. The Japanese synthetics also had the highest concentrations of Ca, S, Cd, and Mo. The US cultivars had concentrations of Ca as high as the Japanese synthetics, and the highest concentrations of Mg and Fe. KASIB’s germplasm had near-average values for most elements. Superior germplasm, with high macro- and microelement concentrations and low trace-element concentrations, was found in all groups of material included.