A diverse set of molecular markers techniques have been developed over the last almost 40 years and used with success for breeding a number of major crops. These have been narrowed down to a few preferred DNA based marker types, and emphasis is now on adapting the technologies to a wide range of crop plants and trees. In this Special Issue, the strength of molecular breeding is revealed through research and review papers that use a combination of molecular markers with other classic breeding techniques to obtain quality improvement of the crop. The constant improvement and maintenance of quality by breeding is crucial and challenged by a changing climate and molecular markers can support the direct introgression of traits into elite breeding lines. All the papers in this Special Issue “Molecular genetics, Genomics, and Biotechnology in Crop Plant Breeding” have attracted significant attention, as can be witnessed by the graphs for each paper on the Journal’s homepage. It is the hope that it will encourage others to use these tools in developing an even wider range of crop plants and trees. Full article

The genetic diversity of 14 Japanese plum (Prunus salicina Lindl) landraces adapted to an ecosystem of alternating flooding and dry conditions was characterized using neutral simple sequence repeat (SSR) markers. Twelve SSRs located in six chromosomes of the Prunus persica reference genome resulted to be polymorphic, thus allowing identification of all the evaluated landraces. Differentiation between individuals was moderate to high (average shared allele distance (DAS) = 0.64), whereas the genetic diversity was high (average indices polymorphism information content (PIC) = 0.62, observed heterozygosity (Ho) = 0.51, unbiased expected heterozygosity (uHe) = 0.70). Clustering and genetic structure approaches grouped all individuals into two major groups that correlated with flesh color. This finding suggests that the intuitive breeding practices of growers tended to select plum trees according to specific phenotypic traits. These neutral markers were adequate for population genetic studies and cultivar identification. Furthermore, we assessed the SSR flanking genome regions (25 kb) in silico to search for candidate genes related to stress resistance or associated with other agronomic traits of interest. Interestingly, at least 26 of the 118 detected genes seem to be related to fruit quality, plant development, and stress resistance. This study suggests that the molecular characterization of specific landraces of Japanese plum that have been adapted to extreme agroecosystems is a useful approach to localize candidate genes which are potentially interesting for breeding. Full article

Restriction site-associated DNA sequencing (RADseq) and its derived protocols, such as double digest RADseq (ddRADseq), offer a flexible and highly cost-effective strategy for efficient plant genome sampling. This has become one of the most popular genotyping approaches for breeding, conservation, and evolution studies in model and non-model plant species. However, universal protocols do not always adapt well to non-model species. Herein, this study reports the development of an optimized and detailed ddRADseq protocol in Eucalyptus dunnii, a non-model species, which combines different aspects of published methodologies. The initial protocol was established using only two samples by selecting the best combination of enzymes and through optimal size selection and simplifying lab procedures. Both single nucleotide polymorphisms (SNPs) and simple sequence repeats (SSRs) were determined with high accuracy after applying stringent bioinformatics settings and quality filters, with and without a reference genome. To scale it up to 24 samples, we added barcoded adapters. We also applied automatic size selection, and therefore obtained an optimal number of loci, the expected SNP locus density, and genome-wide distribution. Reliability and cross-sequencing platform compatibility were verified through dissimilarity coefficients of 0.05 between replicates. To our knowledge, this optimized ddRADseq protocol will allow users to go from the DNA sample to genotyping data in a highly accessible and reproducible way. Full article

Hull opening is a key physiological process during reproductive development, strongly affecting the subsequent fertilization and seed development in rice. In this study, we characterized a rice mutant, non-open hull 1 (noh1), which was derived from ethylmethane-sulfonate (EMS)-treated Xinong 1B (Oryza sativa L.). All the spikelets of noh1 developed elongated and thin lodicules, which caused the failure of hull opening and the cleistogamy. In some spikelets of the noh1, sterile lemmas transformed into hull-like organs. qPCR analysis indicated that the expression of A- and E-function genes was significantly upregulated, while the expression of some B-function genes was downregulated in the lodicules of noh1. In addition, the expression of A-function genes was significantly upregulated, while the expression of some sterile-lemma maker genes was downregulated in the sterile lemma of noh1. These data suggested that the lodicule and sterile lemma in noh1 mutant gained glume-like and lemma-like identity, respectively. Genetic analysis showed that the noh1 trait was controlled by a single recessive gene. The NOH1 gene was mapped between the molecular markers ZJ-9 and ZJ-25 on chromosome 1 with a physical region of 60 kb, which contained nine annotated genes. These results provide a foundation for the cloning and functional research of NOH1 gene. Full article

The phenomenon of cytoplasmic male sterility (CMS), consisting in the inability to produce functional pollen due to mutations in mitochondrial genome, has been described in more than 150 plant species. With the discovery of nuclear fertility restorer (Rf) genes capable of suppressing the CMS phenotype, it became possible to use the CMS-Rf genetic systems as the basis for practical utilization of heterosis effect in various crops. Seed production of sunflower hybrids all over the world is based on the extensive use of the PET1 CMS combined with the Rf1 gene. At the same time, data on Rf1 localization, sequence, and molecular basis for the CMS PET1 type restoration of fertility remain unknown. Searching for candidate genes of the Rf1 gene has great fundamental and practical value. Therefore, in this study, association mapping of fertility restorer gene for CMS PET1 in sunflower was performed. The genome-wide association study (GWAS) results made it possible to isolate a segment 7.72 Mb in length on chromosome 13, in which 21 candidates for Rf1 fertility restorer gene were identified, including 20 pentatricopeptide repeat (PPR)family genes and one Probable aldehyde dehydrogenase gene. The results will serve as a basis for further study of the genetic nature and molecular mechanisms of pollen fertility restoration in sunflower, as well as for further intensification of sunflower breeding. Full article

Development of high yielding and more palatable glutinous rice is an important goal in breeding and long-standing cultural interaction in Asia. In this study, the TGW6 and Wx, major genes conferring 1000 grain weight (GW) and amylose content (AC), were edited in a maintainer line by CRISPR/Cas9 technology. Four targets were assembled in pYLCRISPR/Cas9Pubi-H vector and T₀ mutant plants were obtained through Agrobacterium mediated transformation with 90% mutation frequency having 28% homozygous mutations without off-target effects in three most likely sites of each target and expression level of target genes in mutant lines was significantly decreased (P < 0.01), the GW and gel consistency (GC) were increased, and the AC and gelatinization temperature (GT) were decreased significantly and grain appearance was opaque, while there was no change in starch content (SC) and other agronomic traits. Mutations were inheritable and some T₁ plants were re-edited but T₂ generation was completely stable. The pollen fertility status was randomly distributed, and the mutant maintainer lines were hybridized with Cytoplasmic Male Sterile (CMS) line 209A and after subsequent backcrossing the two glutinous CMS lines were obtained in BC₂F₁. The identified proteins from anthers of CMS and maintainer line were closely associated with transcription, metabolism, signal transduction, and protein biosynthesis. Putative mitochondrial NAD⁺-dependent malic enzyme was absent in CMS line which caused the pollen sterility because of insufficient energy, while upregulation of putative acetyl-CoA synthetase and Isoamylase in both lines might have strong relationship with CMS and amylose content. High yielding glutinous CMS lines will facilitate hybrid rice breeding and investigations of proteins linked to male sterility will provide the insights to complicated metabolic network in anther development. Full article

Drought stress is one of the most adverse environmental limiting factors for wheat (Triticum aestivum L.) productivity worldwide. For better understanding of the molecular mechanism of wheat in response to drought, a comparative transcriptome approach was applied to investigate the gene expression change of two wheat cultivars, Jimai No. 47 (drought-tolerant) and Yanzhan No. 4110 (drought-sensitive) in the field under irrigated and drought-stressed conditions. A total of 3754 and 2325 differential expressed genes (DEGs) were found in Jimai No. 47 and Yanzhan No. 4110, respectively, of which 377 genes were overlapped, which could be considered to be the potential drought-responsive genes. GO (Gene Ontology) analysis showed that these DEGs of tolerant genotype were significantly enriched in signaling transduction and MAP (mitogen-activated protein) kinase activity, while that of sensitive genotype was involved in photosynthesis, membrane protein complex, and guard cell differentiation. Furthermore, 32 and 2 RNA editing sites were identified in drought-tolerant and sensitive genotypes under drought compared to irrigation, demonstrating that RNA editing also plays an important role in response to drought in wheat. This study investigated the gene expression pattern and RNA editing sites of two wheat cultivars with contrasting tolerance in field condition, which will contribute to a better understanding of the molecular mechanism of drought tolerance in wheat and beyond. Full article

Rapeseed (Brassica napus L.) with substantial lipid and oleic acid content is of great interest to rapeseed breeders. Overexpression of Glycine max transcription factors Dof4 and Dof11 increased lipid accumulation in Arabidopsis and microalgae, in addition to modifying the quantity of certain fatty acid components. Here, we report the involvement of GmDof4 and GmDof11 in regulating fatty acid composition in rapeseeds. Overexpression of GmDof4 and GmDof11 in rapeseed increased oleic acid content and reduced linoleic acid and linolenic acid. Both qPCR and the yeast one-hybrid assay indicated that GmDof4 activated the expression of FAB2 by directly binding to the cis-DNA element on its promoters, while GmDof11 directly inhibited the expression of FAD2. Thus, GmDof4 and GmDof11 might modify the oleic acid content in rapeseed by directly regulating the genes that are associated with fatty acid biosynthesis. Full article

Faba bean (Vicia faba L.), a partially allogamous species, is rich in protein. Condensed tannins limit the use of faba beans as food and feed. Two recessive genes, zt-1 and zt-2, control the zero tannin content in faba bean and promote a white flower phenotype. To determine the inheritance and develop a linkage map for the zt-1 gene in the faba bean germplasm M3290, F₂ and F₃ progenies were derived from the purple flower and high tannin content genotypes Qinghai12 and zt-1 line M3290, respectively. Genetic analysis verified a single recessive gene for zero tannin content and flower colour. In total, 596 SSR markers and 100 ISSR markers were used to test the polymorphisms between the parents and bulks for the contrasting flower colour via Bulked Segregant Analysis (BSA). Subsequently, six SSR markers and seven ISSR markers were used to genotype the entire 413 F₂ population. Linkage analysis showed that the zt-1 gene was closely linked to the SSR markers SSR84 and M78, with genetic distances of 2.9 and 5.8 cM, respectively. The two flanked SSR markers were used to test 34 faba bean genotypes with different flower colours. The closely linked SSR marker SSR84 predicted the zt-1 genotypes with absolute accuracy. The results from the marker-assisted selection (MAS) from this study could provide a solid foundation for further faba bean breeding programmes. Full article

In a proof of concept study aimed at showing that metabolites in bread wheat (Triticum aestivum L. ssp. aestivum), phloem exudates have potential as biochemical markers for cultivar discrimination, Argentinean cultivars from three quality groups (groups 1, 2, and 3 of high, intermediate, and low quality, respectively) were grown under two nitrogen (N) availabilities and analysed for metabolic profile by electrospray ionisation mass spectrometry. Data as signal strengths of mass/charge (m/z) values binned to a resolution of 0.2 Daltons were subjected to principal component analysis and orthogonal projections to latent structures discriminant analysis. Certain bins were influential in discriminating groups taken in pairs and some were involved in separating all three groups. In high N availability, group 3 cultivars clustered away from the other cultivars, while group 1 cultivars clustered tightly together; group 2 cultivars were more scattered between group 1 and group 3 cultivars. In low N availability, the cultivars were not clustered as tightly; nonetheless, group 1 cultivars tended to cluster together and mainly separated from those of group 2. m/z values also showed potential for discrimination between N availability. In conclusion, phloem exudate metabolic profiles could provide biochemical markers for selection during breeding and for discerning the effects of N fertiliser application. Full article

Biomass rich in lignocellulose from grasses is a major source for biofuel production and animal feed. However, the presence of lignin in cell walls limits its efficient utilisation such as in its bioconversion to biofuel. Reduction of the lignin content or alteration of its structure in crop plants have been pursued, either by regulating genes encoding enzymes in the lignin biosynthetic pathway using biotechnological techniques or by breeding naturally-occurring low lignin mutant lines. The aim of this review is to provide a summary of these studies, focusing on lignin (monolignol) biosynthesis and composition in grasses and, where possible, the impact on recalcitrance to bioconversion. An overview of transgenic crops of the grass family with regulated gene expression in lignin biosynthesis is presented, including the effect on lignin content and changes in the ratio of p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) units. Furthermore, a survey is provided of low-lignin mutants in grasses, including cereals in particular, summarising their origin and phenotypic traits together with genetics and the molecular function of the various genes identified. Full article

Pre-harvest sprouting (PHS) is one of the most important factors having adverse effects on yield and grain quality all over the world, particularly in wet harvest conditions. PHS is controlled by both genetic and environmental factors and the interaction of these factors. Breeding varieties with high PHS resistance have important implications for reducing yield loss and improving grain quality. The rapid advancements in the wheat genomic database along with transcriptomic and proteomic technologies have broadened our knowledge for understanding the regulatory mechanism of PHS resistance at transcriptomic and post-transcriptomic levels. In this review, we have described in detail the recent advancements on factors influencing PHS resistance, including grain color, seed dormancy, α-amylase activity, plant hormones (especially abscisic acid and gibberellin), and QTL/genes, which are useful for mining new PHS-resistant genes and developing new molecular markers for multi-gene pyramiding breeding of wheat PHS resistance, and understanding the complicated regulatory mechanism of PHS resistance. Full article

Genomic Selection (GS) is a method in plant breeding to predict the genetic value of untested lines based on genome-wide marker data. The method has been widely explored with simulated data and also in real plant breeding programs. However, the optimal strategy and stage for implementation of GS in a plant-breeding program is still uncertain. The accuracy of GS has proven to be affected by the data used in the GS model, including size of the training population, relationships between individuals, marker density, and use of pedigree information. GS is commonly used to predict the additive genetic value of a line, whereas non-additive genetics are often disregarded. In this review, we provide a background knowledge on genomic prediction models used for GS and a view on important considerations concerning data used in these models. We compare within- and across-breeding cycle strategies for implementation of GS in cereal breeding and possibilities for using GS to select untested lines as parents. We further discuss the difference of estimating additive and non-additive genetic values and its usefulness to either select new parents, or new candidate varieties. Full article

Climate change, associated with global warming, extreme weather events, and increasing incidence of weeds, pests and pathogens, is strongly influencing major cropping systems. In this challenging scenario, miscellaneous strategies are needed to expedite the rate of genetic gains with the purpose of developing novel varieties. Large plant breeding populations, efficient high-throughput technologies, big data management tools, and downstream biotechnology and molecular techniques are the pillars on which next generation breeding is based. In this review, we describe the toolbox the breeder has to face the challenges imposed by climate change, remark on the key role bioinformatics plays in the analysis and interpretation of big “omics” data, and acknowledge all the benefits that have been introduced into breeding strategies with the biotechnological and digital revolution. Full article