Yarmilla Reinprecht

In silico comparison of genomic regions containing genes coding for enzymes and transcription factors for the phenylpropanoid pathway in Phaseolus vulgaris L. and Glycine max L. Merr

Legumes contain a variety of phytochemicals derived from the phenylpropanoid pathway that have important effects on human health as well as seed coat color, plant disease resistance and nodulation. However, the information about the genes involved in this important pathway is fragmentary in common bean (Phaseolus vulgaris L.). The objectives of this research were to isolate genes that function in and control the phenylpropanoid pathway in common bean, determine their genomic locations in silico in common bean and soybean, and analyze sequences of the 4CL gene family in two common bean genotypes. Sequences of phenylpropanoid pathway genes available for common bean or other plant species were aligned, and the conserved regions were used to design sequence-specific primers. The PCR products were cloned and sequenced and the gene sequences along with common bean gene-based (g) markers were BLASTed against the Glycine max v.1.0 genome and the P. vulgaris v.1.0 (Andean) early release genome. In addition, gene sequences were BLASTed against the OAC Rex (Mesoamerican) genome sequence assembly. In total, fragments of 46 structural and regulatory phenylpropanoid pathway genes were characterized in this way and placed in silico on common bean and soybean sequence maps. The maps contain over 250 common bean g and SSR (simple sequence repeat) markers and identify the positions of more than 60 additional phenylpropanoid pathway gene sequences, plus the putative locations of seed coat color genes. The majority of cloned phenylpropanoid pathway gene sequences were mapped to one location in the common bean genome but had two positions in soybean. The comparison of the genomic maps confirmed previous studies, which show that common bean and soybean share genomic regions, including those containing phenylpropanoid pathway gene sequences, with conserved synteny. Indels identified in the comparison of Andean and Mesoamerican common bean 4CL gene sequences might be used to develop inter-pool phenylpropanoid pathway gene-based markers. We anticipate that the information obtained by this study will simplify and accelerate selections of common bean with specific phenylpropanoid pathway alleles to increase the contents of beneficial phenylpropanoids in common bean and other legumes.

A comparison of the molecular organization of genomic regions associated with resistance to common bacterial blight in two Phaseolus vulgaris genotypes

Resistance to common bacterial blight, caused by Xanthomonas axonopodis pv. phaseoli, in Phaseolus vulgaris is conditioned by several loci on different chromosomes. Previous studies with OAC-Rex, a CBB-resistant, white bean variety of Mesoamerican origin, identified two resistance loci associated with the molecular markers Pv-CTT001 and SU91, on chromosome 4 and 8, respectively. Resistance to CBB is assumed to be derived from an interspecific cross with Phaseolus acutifolius in the pedigree of OAC-Rex. Our current whole genome sequencing effort with OAC-Rex provided the opportunity to compare its genome in the regions associated with CBB resistance with the v1.0 release of the P. vulgaris line G19833, which is a large seeded bean of Andean origin, and (assumed to be) CBB susceptible. In addition, the genomic regions containing SAP6, a marker associated with P. vulgaris-derived CBB-resistance on chromosome 10, were compared. These analyses indicated that gene content was highly conserved between G19833 and OAC-Rex across the regions examined (>80%). However, fifty-nine genes unique to OAC Rex were identified, with resistance gene homologues making up the largest category (10 genes identified). Two unique genes in OAC-Rex located within the SU91 resistance QTL have homology to P. acutifolius ESTs and may be potential sources of CBB resistance. As the genomic sequence assembly of OAC-Rex is completed, we expect that further comparisons between it and the G19833 genome will lead to a greater understanding of CBB resistance in bean.

Relationships and inheritance of linolenic acid and seed lipoxygenases in soybean crosses designed to combine these traits

To improve oxidative stability of soybean oil and reduce off-flavours, we previously developed low linolenic acid, lipoxygenase-free (LLA.3lx) soybean germplasm. The objectives of this study were to characterize the patterns of inheritance and determine the relationships between the low linolenic acid (LLA) trait derived from the lines RG10 and PI 361088B and seed lipoxygenase nulls (3lx) from a triple null line OX948 that were used to create the new LLA.3lx germplasm. Reciprocal crosses between RG10 and OX948 and between PI 361088B and OX948 were made and populations derived from them were evaluated for their fatty acid profiles and seed lipoxygenases (LX) at the F2, F5 and F6 generations. Both RG10 and PI 361088B contain a single gene that controls linolenic acid (LA) content with alleles acting in an additive manner. No significant cytoplasmic effects were observed on LA content. The LLA trait was highly heritable in both RG10 × OX948 (RO) and PI 361088B × OX948 (PO) crosses and stable in different env...

Microsomal Omega-3 Fatty Acid Desaturase Genes in Low Linolenic Acid Soybean Line RG10 and Validation of Major Linolenic Acid QTL

High levels of linolenic acid (80 g kg−1) are associated with the development of off-flavors and poor stability in soybean oil. The development of low linolenic acid lines such as RG10 (20 g kg−1 linolenic acid) can reduce these problems. The level of linolenic acid in seed oil is determined by the activities of microsomal omega-3 fatty acid desaturases (FAD3). A major linolenic acid QTL (>70% of variation) on linkage group B2 (chromosome Gm14) was previously detected in a recombinant inbred line population from the RG10 × OX948 cross. The objectives of this study were to validate the major linolenic acid QTL in an independent population and characterize all the soybean FAD3 genes. Four FAD3 genes were sequenced and localized in RG10 and OX948 and compared to the genes in the reference Williams 82 genome. The FAD3A gene sequences mapped to the locus Glyma.14g194300 [on the chromosome Gm14 (B2)], which is syntenic to the FAD3B gene (locus Glyma.02g227200) on the chromosome Gm02 (D1b). The location of the FAD3A gene is the same as was previously determined for the fan allele, that conditions low linolenic acid content and several linolenic acid QTL, including Linolen 3-3, mapped previously with the RG10 × OX948 population and confirmed in the PI 361088B × OX948 population as Linolen-PO (FAD3A). The FAD3B gene-based marker, developed previously, was mapped to the chromosome Gm02 (D1b) in a region containing a newly detected linolenic acid QTL [Linolen-RO(FAD3B)] in the RG10 × OX948 genetic map and corresponds well with the in silico position of the FAD3B gene sequences. FAD3C and FAD3D gene sequences, mapped to syntenic regions on chromosomes Gm18 (locus Glyma.18g062000) and Gm11 (locus Glyma.11g227200), respectively. Association of linolenic acid QTL with the desaturase genes FAD3A and FAD3B, their validation in an independent population, and development of FAD3 gene-specific markers should simplify and accelerate breeding for low linolenic acid soybean cultivars.

A Versatile Soybean Recombinant Inbred Line Population Segregating for Low Linolenic Acid and Lipoxygenase Nulls - Molecular Characterization and Utility for Soymilk and Bioproduct Production

The chapter aims to: (a) review the development of a soybean recombinant inbred line (RIL) population obtained from the RG10 x OX948 cross that is segregating for low linolenic acid (LLA) and lipoxygenase nulls (3lx), (b) present the molecular characterization of the LLA and 3lx traits and (c) discuss the applicability of the population for soymilk and biocomposite production. The chapter is organized as follows. Section 2 describes development and evaluation of the RG10 x OX948 RIL population, inheritance of LLA and 3lx traits and the development of novel low linolenic acid, lipoxygenase free (LLA.3lx) germplasm. Section 3 describes the molecular characterization of the LLA and 3lx traits derived from RG10 and OX948, respectively. The versatility of the RIL population for different applications, including soymilk and bioproduct production are presented in Section 4, which illustrates that there is potential for utilizing the whole soybean plant. The conclusion summarizes the results and discusses possible future uses of the RG10 x OX948 RIL population. Problem Oxidation of linolenic acid (LA) is catalyzed by lipoxygenase (LX; linoleate: oxygen oxidoreductase; EC 1.13.11.12) and is associated with off-flavours of soybean products. The hydroperoxides that are produced and their breakdown products impart undesirable grassy-beany and bitter flavours on soybean products (Rackis et al., 1979). Most soybean cultivars contain 70 to 90 g kg-1 LA and approximately 20 g kg-1 of the total seed protein is LX. A possible solution to soybean stability problems is to genetically eliminate seed LX and reduce the content of LA. A number of soybean cultivars with low LA content (LLA; < 40 g kg-1 LA) or seed LX nulls (Hammond et al., 1972; Hildebrand & Hymowitz, 1982; Wilcox, 1985; White, 2000) have been developed. It was shown that oil extracted from LLA lines is more stable than oil extracted from conventional soybean, needs less hydrogenation and consequently, contains less unhealthy trans fatty acids (Mounts et al., 1988), which may reduce the risk of heart disease (Hayakawa et al., 2000). Similarly, protein products from lx3 soybean have more favourable flavour profiles (King et al., 1998). It has been suggested that a combination of seed LX nulls with LLA content might lead to even

Genome Regions Associated with Functional Performance of Soybean Stem Fibers in Polypropylene Thermoplastic Composites

Plant fibers can be used to produce composite materials for automobile parts, thus reducing plastic used in their manufacture, overall vehicle weight and fuel consumption when they replace mineral fillers and glass fibers. Soybean stem residues are, potentially, significant sources of inexpensive, renewable and biodegradable natural fibers, but are not curretly used for biocomposite production due to the functional properties of their fibers in composites being unknown. The current study was initiated to investigate the effects of plant genotype on the performance characteristics of soybean stem fibers when incorporated into a polypropylene (PP) matrix using a selective phenotyping approach. Fibers from 50 lines of a recombinant inbred line population (169 RILs) grown in different environments were incorporated into PP at 20% (wt/wt) by extrusion. Test samples were injection molded and characterized for their mechanical properties. The performance of stem fibers in the composites was significantly affected by genotype and environment. Fibers from different genotypes had significantly different chemical compositions, thus composites prepared with these fibers displayed different physical properties. This study demonstrates that thermoplastic composites with soybean stem-derived fibers have mechanical properties that are equivalent or better than wheat straw fiber composites currently being used for manufacturing interior automotive parts. The addition of soybean stem residues improved flexural, tensile and impact properties of the composites. Furthermore, by linkage and in silico mapping we identified genomic regions to which quantitative trait loci (QTL) for compositional and functional properties of soybean stem fibers in thermoplastic composites, as well as genes for cell wall synthesis, were co-localized. These results may lead to the development of high value uses for soybean stem residue.

A Comparison of Phenylpropanoid Pathway Gene Families in Common Bean. Focus on P450 and C4H Genes

The focus of this chapter is on gene families encoding enzymes of phenylpropanoid pathway in common bean. The introductory section contains a short overview of the phenylpropanoid pathway. Section 11.2 introduces major gene families encoding enzymes of this pathway in common bean, soybean, and Arabidopsis in the current annotations of their complete genome sequences (Phaseolus vulgaris v1.0, Glycine max Wm82.a2.v1, and Arabidopsis thaliana TAIR10) deposited in Phytozome 10.2. For each of the 21 enzyme classes, their functional annotations were based on the commonly used Pfam and KOG databases, while the number of genes in each family was based on Phytozome and KEGG databases. Section 11.3 describes cytochrome P450s involved in the phenylpropanoid pathway with particular emphasis on ten families included in the general (central) phenylpropanoid pathway, C4H (family CYP73A), in the lignin/lignan branch, C3H (family CYP98A) and F5H (family CYP84A), in the flavonoid/anthocyanin/proanthocyanidin branch, F3′H (family CYP75B), F3′5′H (family CYP75A), and FNS (family CYP93B), and in the isoflavonoid branch IFS (family CYP93C), I2′H (family CYP81E), F6H (family CYP71D), and D6aH (family CYP93A). The availability of the complete genome sequences enabled a thorough inventory of putative P450 genes encoding enzymes of this metabolic pathway. The P450 gene sequences from common bean were compared to homologs from Arabidopsis and soybean and confirmed with the information published for both soybean and common bean genomes. Cinnamate 4-hydroxylase (C4H) is the first P450 enzyme in the phenylpropanoid pathway and is described in detail in Sect. 11.4. It belongs to the relatively small CYP73A gene family. Genome locations and gene structures including cis-regulatory regions in 5′UTRs (5′ regulatory sequences) are detailed for this family in common bean. In addition, the expression patterns of these genes in different tissues (Phytozome 10.2) and syntenic relationships (Plant Genome Duplication Database) between common bean and soybean were examined. Finally, genes encoding the C4H enzyme in landrace G19833 (Andean gene pool, Phytozome 10.2) and in cultivar OAC Rex (Mesoamerican gene pool) were compared and searched for polymorphisms. These sequence differences can be used to develop C4H gene-based marker(s) to explore the roles of these genes in various processes such as lignin or anthocyanin biosynthesis.

Technology and Sustainability of Crop Fibre Uses in Bioproducts in Ontario, Canada: Corn Stalk and Cob Fibre Performance in Polypropylene Composites

Composites containing fibres from biological sources, such as residues from field crop production, are being increasingly used for manufacturing consumer products, automobile parts and construction materials because of their low costs, as well as their ecological and performance benefits. The chapter examines the sustainability of using plant fibres for bioproduct manufacturing in Ontario, Canada from annual and perennial crops. It also examines parameters that affect the performance of composites compounded with polypropylene and (Zea mays) corn fibres. In particular, the study identified relationships between specific performance characteristics of the corn fibres and their chemical compositions and confirmed that plant genetics and crop production environment play significant roles in both traits. Further, it identified cell wall traits, genomic regions and genes that might be used to select corn lines that have improved fibre characteristics for bioproduct manufacturing.