Analysis of Genetic Diversity in the Traditional Chinese Medicine Plant 'Kushen' (Sophora flavescens Ait.).

Kushen root, from the woody legume Sophora flavescens, is a traditional Chinese medicine that is a key ingredient in several promising cancer treatments. This activity is attributed in part to two quinolizidine alkaloids (QAs), oxymatrine and matrine, that have a variety of therapeutic activities in vitro. Genetic selection is needed to adapt S. flavescens for cultivation and to improve productivity and product quality. Genetic diversity of S. flavescens was investigated using genotyping-by-sequencing (GBS) on 85 plants grown from seeds collected from 9 provinces of China. DArTSeq provided over 10,000 single nucleotide polymorphism (SNP) markers, 1636 of which were used in phylogenetic analysis to reveal clear regional differences for S. flavescens. One accession from each region was selected for PCR-sequencing to identify gene-specific SNPs in the first two QA pathway genes, lysine decarboxylase (LDC) and copper amine oxidase (CAO). To obtain SfCAO sequence for primer design we used a targeted transcript capture and assembly strategy using publicly available RNA sequencing data. Partial gene sequence analysis of SfCAO revealed two recently duplicated genes, SfCAO1 and SfCAO2, in contrast to the single gene found in the QA-producing legume Lupinus angustifolius. We demonstrate high efficiency converting SNPs to Kompetitive Allele Specific PCR (KASP) markers developing 27 new KASP markers, 17 from DArTSeq data, 7 for SfLDC, and 3 for SfCAO1. To complement this genetic diversity analysis a field trial site has been established in South Australia, providing access to diverse S. flavescens material for morphological, transcriptomic, and QA metabolite analysis. Analysis of dissected flower buds revealed that anthesis occurs before buds fully open suggesting a potential for S. flavescens to be an inbreeding species, however this is not supported by the relatively high level of heterozygosity observed. Two plants from the field trial site were analysed by quantitative real-time PCR and levels of matrine and oxymatrine were assessed in a variety of tissues. We are now in a strong position to select diverse plants for crosses to accelerate the process of genetic selection needed to adapt kushen to cultivation and improve productivity and product quality.

The barley pan-genome reveals the hidden legacy of mutation breeding.

Genetic diversity is key to crop improvement. Owing to pervasive genomic structural variation, a single reference genome assembly cannot capture the full complement of sequence diversity of a crop species (known as the 'pan-genome'1). Multiple high-quality sequence assemblies are an indispensable component of a pan-genome infrastructure. Barley (Hordeum vulgare L.) is an important cereal crop with a long history of cultivation that is adapted to a wide range of agro-climatic conditions2. Here we report the construction of chromosome-scale sequence assemblies for the genotypes of 20 varieties of barley-comprising landraces, cultivars and a wild barley-that were selected as representatives of global barley diversity. We catalogued genomic presence/absence variants and explored the use of structural variants for quantitative genetic analysis through whole-genome shotgun sequencing of 300 gene bank accessions. We discovered abundant large inversion polymorphisms and analysed in detail two inversions that are frequently found in current elite barley germplasm; one is probably the product of mutation breeding and the other is tightly linked to a locus that is involved in the expansion of geographical range. This first-generation barley pan-genome makes previously hidden genetic variation accessible to genetic studies and breeding.

A GDSL Esterase/Lipase Catalyzes the Esterification of Lutein in Bread Wheat.

Xanthophylls are a class of carotenoids that are important micronutrients for humans. They are often found esterified with fatty acids in fruits, vegetables, and certain grains, including bread wheat (Triticum aestivum). Esterification promotes the sequestration and accumulation of carotenoids, thereby enhancing stability, particularly in tissues such as in harvested wheat grain. Here, we report on a plant xanthophyll acyltransferase (XAT) that is both necessary and sufficient for xanthophyll esterification in bread wheat grain. XAT contains a canonical Gly-Asp-Ser-Leu (GDSL) motif and is encoded by a member of the GDSL esterase/lipase gene family. Genetic evidence from allelic variants of wheat and transgenic rice (Oryza sativa) calli demonstrated that XAT catalyzes the formation of xanthophyll esters. XAT has broad substrate specificity and can esterify lutein, ß-cryptoxanthin, and zeaxanthin using multiple acyl donors, yet it has a preference for triacylglycerides, indicating that the enzyme acts via transesterification. A conserved amino acid, Ser-37, is required for activity. Despite xanthophylls being synthesized in plastids, XAT accumulated in the apoplast. Based on analysis of substrate preferences and xanthophyll ester formation in vitro and in vivo using xanthophyll-accumulating rice callus, we propose that disintegration of the cellular structure during wheat grain desiccation facilitates access to lutein-promoting transesterification.plantcell;31/12/3092/FX1F1fx1.

Fine mapping of Rha2 in barley reveals candidate genes for resistance against cereal cyst nematode.

KEY MESSAGE: The cereal cyst nematode resistance locus Rha2 was mapped to a 978 kbp region on the long arm of barley chromosome 2H. Three candidate genes are discussed. The cereal cyst nematode (CCN) Heterodera avenae is a soil-borne obligate parasite that can cause severe damage to cereals. This research involved fine mapping of Rha2, a CCN resistance locus on chromosome 2H of barley. Rha2 was previously mapped relative to restriction fragment length polymorphisms (RFLPs) in two mapping populations. Anchoring of flanking RFLP clone sequences to the barley genome assembly defined an interval of 5077 kbp. Genotyping-by-sequencing of resistant and susceptible materials led to the discovery of potentially useful single nucleotide polymorphisms (SNPs). Assays were designed for these SNPs and applied to mapping populations. This narrowed the region of interest to 3966 kbp. Further fine mapping was pursued by crossing and backcrossing the resistant cultivar Sloop SA to its susceptible ancestor Sloop. Evaluation of F2 progeny confirmed that the resistance segregates as a single dominant gene. Genotyping of 9003 BC2F2 progeny identified recombinants. Evaluation of recombinant BC2F3 progeny narrowed the region of interest to 978 kbp. Two of the SNPs within this region proved to be diagnostic of CCN resistance across a wide range of barley germplasm. Fluorescence-based and gel-based assays were developed for these SNPs for use in marker-assisted selection. Within the candidate region of the reference genome, there are nine high-confidence predicted genes. Three of these, one that encodes RAR1 (a cysteine- and histidine-rich domain-containing protein), one that is predicted to encode an acetylglutamate kinase and one that is predicted to encode a tonoplast intrinsic protein, are discussed as candidate genes for CCN resistance.

A locus on barley chromosome 5H affects adult plant resistance to powdery mildew.

Adult plant resistance against plant pathogens is of interest as a means to achieve durable resistance. Prior to this research, the barley lines CLE210 (from Uruguay) and Denar (from the Czech Republic) had been reported to exhibit adult-plant resistance against powdery mildew. Here, populations of doubled haploid lines from crosses of these lines with the susceptible cultivar Baudin were evaluated for powdery mildew resistance in field experiments. Using linkage maps constructed from genotyping-by-sequencing (GBS) data, it was determined that differences in resistance were largely attributable to a region on the long arm of chromosome 5H (5HL). Therefore, KASP™ assays were developed based on GBS tag sequences mapped on that chromosome, providing more reliable genetic maps. In each population, a large-effect QTL was mapped on 5HL. As no sequence variation was detected between CLE210 and Denar in this region of 5HL, the two sources of resistance may be identical by descent in the QTL region and carry the same resistance gene. Marker assays from the QTL region were evaluated on a panel of barley lines, providing information that breeders could use to select assays for use in marker-assisted selection.

A second 'overexpression' allele at the Glu-B1 high-molecular-weight glutenin locus of wheat: sequence characterisation and functional effects.

Bread is one of the major constituents of the human diet and wheat (Triticum aestivum L.) is the most important cereal for bread making. The gluten proteins (glutenins and gliadins) are recognised as important components affecting the processing quality of wheat flour. In this research, we investigated a particular glutenin subunit allele in an Australian cultivar, H45. Based on protein and DNA assays, the Glu-B1 allele of H45 seems to be Glu-B1al, an allele that includes a functional duplication of a gene encoding an x-type high-molecular-weight glutenin subunit, and is thought to increase dough strength through overexpression of that subunit. Yet H45 does not have the dough properties that would be expected if it carries the Glu-B1al allele. After confirming that H45 overexpresses Bx subunits and that it has relatively low un-extractable polymeric protein (an indicator of weak dough), we cloned and sequenced two Bx genes from H45. The sequences of the two genes differ from each other, and they each differ by four single-nucleotide polymorphisms (SNPs) from the sequence that has been reported for the Glu-B1al x-type glutenin genes of the Canadian wheat cultivar Glenlea. One of the SNPs leads to an extra cysteine residue in one of the subunits. The presence of this additional cysteine may explain the dough properties of H45 through effects on cross-linkage within or between glutenin subunits. We propose that the Glu-B1 allele of H45 be designated Glu-B1br, and we present evidence that Glu-B1br is co-inherited with low un-extractable polymeric protein.

HvNax3--a locus controlling shoot sodium exclusion derived from wild barley (Hordeum vulgare ssp. spontaneum).

Previous work identified the wild barley (Hordeum vulgare ssp. spontaneum) accession CPI-71284-48 as being capable of limiting sodium (Na(+)) accumulation in the shoots under saline hydroponic growth conditions. Quantitative trait locus (QTL) analysis using a cross between CPI-71284-48 and a selection of the cultivated barley (H. vulgare ssp. vulgare) cultivar Barque (Barque-73, a moderate Na(+) excluder) attributed the control of the Na(+) exclusion trait from CPI-71284-48 to a single locus on the short arm of chromosome 7H, which was named HvNax3. The locus reduced shoot Na(+) accumulation by 10-25% in plants grown in 150 mM NaCl. Markers generated using colinearity with rice and Brachypodium, together with the analysis of introgression lines and F(2) and F(3) families, enabled HvNax3 to be mapped to a 1.3-cM interval. Genes from the corresponding rice and Brachypodium intervals encode 16 different classes of proteins and include several plausible candidates for HvNax3. The potential of HvNax3 to provide a useful trait contributing to salinity tolerance in cultivated barley is discussed.

Multiplex-ready PCR: a new method for multiplexed SSR and SNP genotyping.

BACKGROUND: Microsatellite (SSR) and single nucleotide polymorphism (SNP) markers are widely used in plant breeding and genomic research. Thus, methods to improve the speed and efficiency of SSR and SNP genotyping are highly desirable. Here we describe a new method for multiplex PCR that facilitates fluorescence-based SSR genotyping and the multiplexed preparation of DNA templates for SNP assays. RESULTS: We show that multiplex-ready PCR can achieve a high (92%) success rate for the amplification of published sequences under standardised reaction conditions, with a PCR specificity comparable to that of conventional PCR methods. We also demonstrate that multiplex-ready PCR supports an improved level of multiplexing in plant genomes of varying size and ploidy, without the need to carefully optimize assay conditions. Several advantages of multiplex-ready PCR for SSR and SNP genotyping are demonstrated and discussed. These include the uniform amplification of target sequences within multiplexed reactions and between independent assays, and the ability to label amplicons during PCR with specialised moieties such fluorescent dyes and biotin. CONCLUSION: Multiplex-ready PCR provides several technological advantages that can facilitate fluorescence-based SSR genotyping and the multiplexed preparation of DNA templates for SNP assays. These advantages can be captured at several points in the genotyping process, and offer considerable cost and labour savings. Multiplex-ready PCR is broadly applicable to plant genomics and marker assisted breeding, and should be transferable to any animal or plant species.