A toolkit to rapidly modify root systems through single plant selection

Background The incorporation of root traits into elite germplasm is typically a slow process. Thus, innovative approaches are required to accelerate research and pre-breeding programs targeting root traits to improve yield stability in different environments and soil types. Marker-assisted selection (MAS) can help to speed up the process by selecting key genes or quantitative trait loci (QTL) associated with root traits. However, this approach is limited due to the complex genetic control of root traits and the limited number of well-characterised large effect QTL. Coupling MAS with phenotyping could increase the reliability of selection. Here we present a useful framework to rapidly modify root traits in elite germplasm. In this wheat exemplar, a single plant selection (SPS) approach combined three main elements: phenotypic selection (in this case for seminal root angle); MAS using KASP markers (targeting a root biomass QTL); and speed breeding to accelerate each cycle. Results To develop a SPS approach that integrates non-destructive screening for seminal root angle and root biomass, two initial experiments were conducted. Firstly, we demonstrated that transplanting wheat seedlings from clear pots (for seminal root angle assessment) into sand pots (for root biomass assessment) did not impact the ability to differentiate genotypes with high and low root biomass. Secondly, we demonstrated that visual scores for root biomass were correlated with root dry weight (r = 0.72), indicating that single plants could be evaluated for root biomass in a non-destructive manner. To highlight the potential of the approach, we applied SPS in a backcrossing program which integrated MAS and speed breeding for the purpose of rapidly modifying the root system of elite bread wheat line Borlaug100. Bi-directional selection for root angle in segregating generations successfully shifted the mean root angle by 30° in the subsequent generation (P ≤ 0.05). Within 18 months, BC2F4:F5 introgression lines were developed that displayed a full range of root configurations, while retaining similar above-ground traits to the recurrent parent. Notably, the seminal root angle displayed by introgression lines varied more than 30° compared to the recurrent parent, resulting in lines with both narrow and wide root angles, and high and low root biomass phenotypes. Conclusion The SPS approach enables researchers and plant breeders to rapidly manipulate root traits of future crop varieties, which could help improve productivity in the face of increasing environmental fluctuations. The newly developed elite wheat lines with modified root traits provide valuable materials to study the value of different root systems to support yield in different environments and soil types.

Expanding the Benefits of Tnt1 for the Identification of Dominant Mutations in Polyploid Crops: A Single Allelic Mutation in the MsNAC39 Gene Produces Multifoliated Alfalfa

Most major crops are polyploid species and the production of genetically engineered cultivars normally requires the introgression of transgenic or gene-edited traits into elite germplasm. Thus, a main goal of plant research is the search of systems to identify dominant mutations. In this article, we show that the Tnt1 element can be used to identify dominant mutations in allogamous tetraploid cultivated alfalfa. Specifically, we show that a single allelic mutation in the MsNAC39 gene produces multifoliate leaves (mfl) alfalfa plants, a pivot trait of breeding programs of this forage species. Finally, we discuss the potential application of a combination of preliminary screening of beneficial dominant mutants using Tnt1 mutant libraries and genome editing via the CRISPR/Cas9 system to identify target genes and to rapidly improve both autogamous and allogamous polyploid crops.

Identification of elite germplasm of medicinally important Andrographis paniculata (Burm. f.) Nees with high content of four active diterpenoids in aerial parts from wild populations of eastern India

Andrographis paniculata is an Indian medicinal plant with tremendous therapeutic values due to the presence of active diterpenoids in its aerial parts. However, high domestic and export demand has led to overexploitation of wild populations of this species. With a view to bringing A. paniculata into cultivation and to reduce the pressure on wild populations, the present study was undertaken to identify elite germplasm from different locations of eastern India by analysing intraspecific variation in the content of four major active diterpenoids. A total of 166 wild accessions of A. paniculata analysed through high-performance liquid chromatography (HPLC) revealed remarkable variation in the sum of four active diterpenoids in the aerial parts, ranging from 0.41 to 8.55% on a dry weight basis. Three elite accessions (AP-6, AP-8, AP-46) having respectively 8.02, 8.36 & 8.55% of the sum of four major active diterpenoids were identified. These germplasm could be used for commercial cultivation and genetic improvement of A. paniculata.

Occurrence of Macrophomina phaseolina in Israel: Challenges for Disease Management and Crop Germplasm Enhancement

Macrophomina phaseolina is a soil-borne fungal pathogen infecting many important crop plants. The fungus, which can survive on crop debris for a long period of time, causes charcoal rot disease by secreting a diverse array of cell-wall degrading enzymes and toxins. M. phaseolina thrives during periods of high temperatures and arid conditions, as typically occuring in Israel and other countries with a Mediterranean climate. Crop losses due to charcoal rot can be expected to increase and spread to other countries in a warming global climate. Management of this pathogen is challenging, requiring an array of approaches for the various crop hosts. Approaches that have had some success in Israel include grafting of melons and watermelons on resistant squash rootstocks and soil application of fungicide to reduce disease incidence in melons, fumigation and alterations in planting date and mulching of strawberries, and alteration in irrigation regime of cotton. Elsewhere, these approaches as well as soil amendments, and addition of organisms that are antagonistic to M. phaseolina have had success in some crop situations. Management through host resistance would be the most sustainable approach, but requires identifying resistant germplasm for each crop and introgressing the resistance into the leading cultivars. Resistance to charcoal rot is under complex genetic control in most crops, posing a great challenge for its introgression into elite germplasm. Moreover, fast, reliable methods of screening for resistance would have to be developed for each crop. The toothpick-inoculation method used by us holds great promise for selecting resistant germplasm for melons and possibly for sesame, but other methodologies have to be devised for each individual crop.

Introducing Beneficial Alleles from Plant Genetic Resources into the Wheat Germplasm

Wheat (Triticum sp.) is one of the world’s most important crops, and constantly increasing its productivity is crucial to the livelihoods of millions of people. However, more than a century of intensive breeding and selection processes have eroded genetic diversity in the elite genepool, making new genetic gains difficult. Therefore, the need to introduce novel genetic diversity into modern wheat has become increasingly important. This review provides an overview of the plant genetic resources (PGR) available for wheat. We describe the most important taxonomic and phylogenetic relationships of these PGR to guide their use in wheat breeding. In addition, we present the status of the use of some of these resources in wheat breeding programs. We propose several introgression schemes that allow the transfer of qualitative and quantitative alleles from PGR into elite germplasm. With this in mind, we propose the use of a stage-gate approach to align the pre-breeding with main breeding programs to meet the needs of breeders, farmers, and end-users. Overall, this review provides a clear starting point to guide the introgression of useful alleles over the next decade.

A Toolkit to Rapidly Modify Root Systems Through Single Plant Selection

Background: The incorporation of root traits into elite germplasm is typically a slow process. Thus, innovative approaches are required to accelerate research and pre-breeding programs targeting root traits to improve yield stability in different environments and soil types. Marker-assisted selection (MAS) can help to speed up the process by selecting key genes or quantitative trait loci (QTL) associated with root traits. However, this approach is limited due to the complex genetic control of root traits and the limited number of well-characterised large effect QTL. Coupling MAS with phenotyping could increase the reliability of selection. Here we present a useful framework to rapidly modify root traits in elite germplasm. In this wheat exemplar, a single plant selection (SPS) approach combined three main elements: phenotypic selection (in this case for seminal root angle); MAS using KASP markers (targeting a root biomass QTL); and speed breeding to accelerate each cycle.Results: To develop a SPS approach that integrates non-destructive screening for seminal root angle and root biomass, two initial experiments were conducted. Firstly, we demonstrated that transplanting wheat seedlings from clear pots (for seminal root angle assessment) into sand pots (for root biomass assessment) did not impact the ability to differentiate genotypes with high and low root biomass. Secondly, we demonstrated that visual scores for root biomass were correlated with root dry weight (r = 0.73), indicating that single plants could be evaluated for root biomass in a non-destructive manner. To highlight the potential of the approach, we applied SPS in a backcrossing program which integrated MAS and speed breeding for the purpose of rapidly modifying the root system of elite bread wheat line Borlaug100. Bi-directional selection for root angle in segregating generations successfully shifted the mean root angle by 30o in the subsequent generation (P ≤ 0.05). Within 18 months, BC2F4:F5 introgression lines were developed that displayed a full range of root configurations, while retaining similar above-ground traits to the recurrent parent. Notably, the seminal root angle displayed by introgression lines varied more than 30° compared to the recurrent parent, resulting in lines with both narrow and wide root angles, and high and low root biomass phenotypes.Conclusion: The SPS approach enables researchers and plant breeders to rapidly manipulate root traits of future crop varieties, which could help improve productivity in the face of increasing environmental fluctuations. The newly developed elite wheat lines with modified root traits provide valuable materials to study the value of different root systems to support yield in different environments and soil types.

Usefulness of Adapted Exotic Maize Lines Developed By Doubled Haploid and Single Seed Descent Methods

Adapted exotic maize (Zea mays L.) germplasm, such as BS39, provides a unique opportunity for broadening the genetic base of U.S. Corn Belt germplasm. In vivo doubled haploid (DH) technology has been used to efficiently exploit exotic germplasm. It can help to purge deleterious recessive alleles. The objectives of this study were to determine the usefulness of BS39-derived inbred lines using both SSD and DH methods, to determine the impact of spontaneous as compared to artificial haploid genome doubling on genetic variance among BS39-derived DH lines, and to identify SNP markers associated with agronomic traits among BS39 inbreds monitored at testcross level. We developed two sets of inbred lines directly from BS39 by DH and SSD methods, named BS39_DH and BS39_SSD. Additionally, two sets were derived from a cross between BS39 and A427 (SHGD donor) by DH and SSD methods, named BS39×A427_DH and BS39×A427_SSD, respectively. Grain yield, moisture, plant height, ear height, stalk lodging, and root lodging were measured to estimate genetic parameters. For genome-wide association (GWAS) analysis, inbred lines were genotyped using Genotype-by-Sequencing (GBS) and Diversity Array Technology Sequencing (DArTSeq). Some BS39-derived inbred lines performed better than elite germplasm inbreds and all sets showed significant genetic variance. The presence of spontaneous haploid genome doubling genes did not affect performance of inbred lines. Five SNPs were significant and three of them located within genes related to plant development or abiotic stresses. These results demonstrate the potential of BS39 to add novel alleles to temperate elite germplasm.

Genetic Diversity in Local and Exotic Avena sativa L. (Oat) Germplasm Using Multivariate Analysis

Avena sativa L., also known as Oat belongs to the Poaceae family, is one of the most significant crops that is grown for its seeds, fodder as well as for human consumption as oatmeal. In the current study, 236 genotypes of A. sativa were analysed for genetic diversity through agro-morphological and SDS-PAGE analysis. Cluster analysis based on agro-morphological characteristics grouped all the genotypes into nine clusters, whereas genotype numbers 537 and 728 were highly different from others. The seed yield production of cluster 9 genotypes was the highest per plant (38.2 ± 0.20 g), while cluster 2 genotypes produced maximum biomass per plant (122.5 ± 9.55 g) as compared to other clusters. In a principal component analysis where four variables were studied, and the observed total variations were 57.60%. Among the genotypes, a maximum grain yield of 38.2 g (each) was recorded for genotypes 22,350 and 728, followed by genotypes 737 and 22,390 (with 36.4 g and 35.6 g of seed productions, respectively). The SDS-PAGE analysis resulted in 13 bands and all the genotypes were grouped into seventeen clusters. At the extreme periphery of the dendrogram, genotype 537 and 22,332 were considered to be the most diverse genotypes. Our findings have implications for both understanding the diversity and relationships among these diverse genotypes of A. sativa and will provide a basis for obtaining the elite germplasm optimally adapted to local conditions. The selected genotypes based on agronomic performance may be potential breeding material to raise successful future cultivars.

Genetic diversity of local and introduced cassava germplasm in Burundi using DArTseq molecular analyses

In Burundi, most of small-scale farmers still grow traditional cassava landraces that are adapted to local conditions and have been selected for consumer preferred attributes. They tend to be susceptible, in varying degrees, to devastating cassava viral diseases such as Cassava Brown Steak Disease (CBSD) and Cassava Mosaic Disease (CMD) with production annual losses of US$1 billion annually. For long term resistance to the disease, several breeding strategies have been proposed. A sound basis for a breeding program is to understand the genetic diversity of both landraces and elite introduced breeding cultivars. This will also assist in efforts to conserve landraces ahead of the broad distribution of improved varieties which have the possibility of replacing landraces. Our study aimed at determining the genetic diversity and relationships of local landraces and introduced elite germplasm using morphological and simple nucleotide polymorphism (SNP) markers as well as identifying a core set of germplasm from the local varieties to be used in the cassava breeding program. A total of 118 cultivars were characterized for morphological trait variation based on leaf, stem and root traits, and genetic variation using SNP markers. Results of morphological characterization based on Ward’s Method revealed three main clusters and five accessions sharing similar characteristics. Molecular characterization identified over 18,000 SNPs and six main clusters and three pairs of duplicates which should be pooled together as one cultivar to avoid redundancy. Results of population genetic analysis showed low genetic distance between populations and between local landraces and elite germplasm. Accessions that shared similar morphological traits were divergent at the molecular level indicating that clustering using morphological traits was inconsistent. Despite the variabilities found within the collection, it was observed that cassava germplasm in Burundi have a narrow genetic base.