Chengdao Li

A chromosome conformation capture ordered sequence of the barley genome

Cereal grasses of the Triticeae tribe have been the major food source in temperate regions since the dawn of agriculture. Their large genomes are characterized by a high content of repetitive elements and large pericentromeric regions that are virtually devoid of meiotic recombination. Here we present a high-quality reference genome assembly for barley (Hordeum vulgare L.). We use chromosome conformation capture mapping to derive the linear order of sequences across the pericentromeric space and to investigate the spatial organization of chromatin in the nucleus at megabase resolution. The composition of genes and repetitive elements differs between distal and proximal regions. Gene family analyses reveal lineage-specific duplications of genes involved in the transport of nutrients to developing seeds and the mobilization of carbohydrates in grains. We demonstrate the importance of the barley reference sequence for breeding by inspecting the genomic partitioning of sequence variation in modern elite germplasm, highlighting regions vulnerable to genetic erosion.

The development of oat microsatellite markers and their use in identifying relationships among Avena species and oat cultivars

Abstract Microsatellites have many desirable marker properties. There has been no report of the development and utilization of microsatellite markers in oat. The objectives of the present study were to construct oat microsatellite-enriched libraries, to isolate microsatellite sequences and evaluate their level of polymorphism in Avena species and oat cultivars. One hundred clones were isolated and sequenced from three oat microsatellite-libraries enriched for either (AC/TG)n, (AG/TC)n or (AAG/TTC)n repeats. Seventy eight clones contained microsatellites. A database search showed that 42% of the microsatellite flanking sequences shared significant homology with various repetitive elements. Alu and retrotransposon sequences were the two largest groups associated with the microsatellites. Forty four primer sets were used to amplify the DNA from 12 Avena species and 20 Avena sativa cultivars. Sixty two percent of the primers revealed polymorphism among the Avena species, but only 36% among the cultivars. In the cultivars, the microsatellites associated with repetitive elements were less polymorphic than those not associated with repetitive elements. Only 25% of the microsatellites associated with repetitive elements were polymorphic, while 46% of the microsatellites not associated with repetitive elements showed polymorphism in the cultivars. An average of four alleles with a polymorphism information content (PIC) of 0.57 per primer set was detected among the Avena species, and 3.8 alleles with a PIC of 0.55 among the cultivars. In addition, 54 barley microsatellite primers were tested in Avena species and 26% of the primers amplified microsatellites from oat. Using microsatellite polymorphisms, dendrograms were constructed showing phylogenetic relationships among Avena species and genetic relationships among oat cultivars.

Molecular basis of barley quality

The quality of barley for the range of end uses from animal feed to brewing is determined by many genes, making the breeding of new barley varieties difficult. Understanding of the molecular basis of barley quality has been advanced by biochemical studies. More recently, molecular genetic tools are allowing the analysis of the biochemical factors contributing to grain quality. Many genetic loci influencing key quality attributes have been identified by gene mapping. Limited success has been reported in using this information to select for quantitative trait loci for these quality traits in plant breeding. Genomic techniques allowing more detailed analysis of variations in the barley genome in relation to quality promise to extend significantly the value of molecular genetic approaches to barley quality improvement. Definition of the genetic basis of malting quality requires the identification of the genes involved in germination and endosperm modification. Feed quality remains difficult to define. Recent advances are likely to accelerate the rate of discovery, providing new options for analysis of barley quality.

Genes controlling seed dormancy and pre-harvest sprouting in a rice-wheat-barley comparison.

Pre-harvest sprouting results in significant economic loss for the grain industry around the world. Lack of adequate seed dormancy is the major reason for pre-harvest sprouting in the field under wet weather conditions. Although this trait is governed by multiple genes it is also highly heritable. A major QTL controlling both pre-harvest sprouting and seed dormancy has been identified on the long arm of barley chromosome 5H, and it explains over 70% of the phenotypic variation. Comparative genomics approaches among barley, wheat and rice were used to identify candidate gene(s) controlling seed dormancy and hence one aspect of pre-harvest sprouting. The barley seed dormancy/pre-harvest sprouting QTL was located in a region that showed good synteny with the terminal end of the long arm of rice chromosome 3. The rice DNA sequences were annotated and a gene encoding GA20-oxidase was identified as a candidate gene controlling the seed dormancy/pre-harvest sprouting QTL on 5HL. This chromosomal region also shared synteny with the telomere region of wheat chromosome 4AL, but was located outside of the QTL reported for seed dormancy in wheat. The wheat chromosome 4AL QTL region for seed dormancy was syntenic to both rice chromosome 3 and 11. In both cases, corresponding QTLs for seed dormancy have been mapped in rice.

Comparative Proteomic Analysis of Aluminum Tolerance in Tibetan Wild and Cultivated Barleys

Aluminum (Al) toxicity is a major limiting factor for plant production in acid soils. Wild barley germplasm is rich in genetic diversity and may provide elite genes for crop Al tolerance improvement. The hydroponic-experiments were performed to compare proteomic and transcriptional characteristics of two contrasting Tibetan wild barley genotypes Al- resistant/tolerant XZ16 and Al-sensitive XZ61 as well as Al-resistant cv. Dayton. Results showed that XZ16 had less Al uptake and translocation than XZ61 and Dayton under Al stress. Thirty-five Al-tolerance/resistance-associated proteins were identified and categorized mainly in metabolism, energy, cell growth/division, protein biosynthesis, protein destination/storage, transporter, signal transduction, disease/defense, etc. Among them, 30 were mapped on barley genome, with 16 proteins being exclusively up-regulated by Al stress in XZ16, including 4 proteins (S-adenosylmethionine-synthase 3, ATP synthase beta subunit, triosephosphate isomerase, Bp2A) specifically expressed in XZ16 but not Dayton. The findings highlighted the significance of specific-proteins associated with Al tolerance, and verified Tibetan wild barley as a novel genetic resource for Al tolerance.

GA-20 oxidase as a candidate for the semidwarf gene sdw1/denso in barley.

The barley sdw1/denso gene not only controls plant height but also yield and quality. The sdw1/denso gene was mapped to the long arm of chromosome 3H. Comparative genomic analysis revealed that the sdw1/denso gene was located in the syntenic region of the rice semidwarf gene sd1 on chromosome 1. The sd1 gene encodes a gibberellic acid (GA)-20 oxidase enzyme. The gene ortholog of rice sd1 was isolated from barley using polymerase chain reaction. The barley and rice genes showed a similar gene structure consisting of three exons and two introns. Both genes share 88.3% genomic sequence similarity and 89% amino acid sequence identity. A single nucleotide polymorphism was identified in intron 2 between barley varieties Baudin and AC Metcalfe with Baudin known to contain the denso semidwarf gene. The single nucleotide polymorphism (SNP) marker was mapped to chromosome 3H in a doubled haploid population of Baudin × AC Metcalfe with 178 DH lines. Quantitative trait locus analysis revealed that plant height cosegregated with the SNP. The sdw1/denso gene in barley is the most likely ortholog of the sd1 in rice. The result will facilitate understanding of the molecular mechanism controlling semidwarf phenotype and provide a diagnostic marker for selection of semidwarf gene in barley.

Application of next-generation sequencing for rapid marker development in molecular plant breeding: a case study on anthracnose disease resistance in Lupinus angustifolius L.

BackgroundIn the last 30 years, a number of DNA fingerprinting methods such as RFLP, RAPD, AFLP, SSR, DArT, have been extensively used in marker development for molecular plant breeding. However, it remains a daunting task to identify highly polymorphic and closely linked molecular markers for a target trait for molecular marker-assisted selection. The next-generation sequencing (NGS) technology is far more powerful than any existing generic DNA fingerprinting methods in generating DNA markers. In this study, we employed a grain legume crop Lupinus angustifolius (lupin) as a test case, and examined the utility of an NGS-based method of RAD (restriction-site associated DNA) sequencing as DNA fingerprinting for rapid, cost-effective marker development tagging a disease resistance gene for molecular breeding.ResultsTwenty informative plants from a cross of RxS (disease resistant x susceptible) in lupin were subjected to RAD single-end sequencing by multiplex identifiers. The entire RAD sequencing products were resolved in two lanes of the 16-lanes per run sequencing platform Solexa HiSeq2000. A total of 185 million raw reads, approximately 17 Gb of sequencing data, were collected. Sequence comparison among the 20 test plants discovered 8207 SNP markers. Filtration of DNA sequencing data with marker identification parameters resulted in the discovery of 38 molecular markers linked to the disease resistance gene Lanr1. Five randomly selected markers were converted into cost-effective, simple PCR-based markers. Linkage analysis using marker genotyping data and disease resistance phenotyping data on a F8 population consisting of 186 individual plants confirmed that all these five markers were linked to the R gene. Two of these newly developed sequence-specific PCR markers, AnSeq3 and AnSeq4, flanked the target R gene at a genetic distance of 0.9 centiMorgan (cM), and are now replacing the markers previously developed by a traditional DNA fingerprinting method for marker-assisted selection in the Australian national lupin breeding program.ConclusionsWe demonstrated that more than 30 molecular markers linked to a target gene of agronomic trait of interest can be identified from a small portion (1/8) of one sequencing run on HiSeq2000 by applying NGS based RAD sequencing in marker development. The markers developed by the strategy described in this study are all co-dominant SNP markers, which can readily be converted into high throughput multiplex format or low-cost, simple PCR-based markers desirable for large scale marker implementation in plant breeding programs. The high density and closely linked molecular markers associated with a target trait help to overcome a major bottleneck for implementation of molecular markers on a wide range of germplasm in breeding programs. We conclude that application of NGS based RAD sequencing as DNA fingerprinting is a very rapid and cost-effective strategy for marker development in molecular plant breeding. The strategy does not require any prior genome knowledge or molecular information for the species under investigation, and it is applicable to other plant species.

Mapping and validation of the genes for resistance to Pyrenophora teres f. teres in barley (Hordeum vulgare L.)

Identification and deployment of disease resistance genes are key objectives of Australian barley breeding programs. Two doubled haploid (DH) populations derived from Tallon × Kaputar (TK) and VB9524 × ND11231 (VN) crosses were used to identify markers for net type net blotch (NTNB) (Pyrenophora teres f. teres). The maps included 263 and 250 markers for TK and VN populations, respectively. The TK population was screened with 5 pathotypes and the VN population with 1 pathotype of NTNB as seedlings in the glasshouse. In addition, the TK population was subjected to natural infection in the field at Hermitage Research Station, Qld. Analyses of the markers were performed using the software packages MapManager and Qgene. One region on chromosome 6H was strongly associated with resistance to NTNB in both populations (R2 = 83% for TK and 66% for VN). In the TK population, 2 more quantitative trait loci (QTLs) were identified on chromosomes 2H and 3H, with R2 values of 30% and 31%, respectively. These associations were consistent over all pathotypes studied during the seedling stage. The same QTL on chromosome 6H was also found to be highly significantly associated (R2 = 65%) with the adult plant (field) response in the TK population. There are several very closely linked markers showing strong associations in these regions. Association of the 4 markers on chromosome 6H QTL with resistance to the NTNB has been validated in 2 other DH populations derived from barley crosses Pompadour × Stirling and WPG8412 × Stirling. These markers present an opportunity for marker assisted selection of lines resistant to NTNB in barley breeding programs.

Identification of QTLs associated with salinity tolerance at late growth stage in barley

Salinity is a major abiotic stress to barley (Hordum vulgare L.) growth and yield. In the current study, quantitative trait loci (QTL) for yield and physiological components at the late growth stage under salt stress and non-stress environments were determined in barley using a double haploid population derived from a cross between CM72 (salt-tolerant) and Gairdner (salt-sensitive). A total of 30 QTLs for 10 traits, including tiller numbers (TN), plant height, spikes per line (SPL), spikes per plant (SPP), dry weight per plant, grains per plant, grain yield, shoot Na+ (NA) and K+ concentraitions (K) in shoot, and Na+/K+ ratio (NAK), were detected, with 17 and 13 QTLs under non-stress and salt stress, respectively. The phenotypic variation explained by individual QTL ranged from 3.25 to 29.81%. QTL flanked by markers bPb-1278 and bPb-8437 on chromosomes 4H was associated with TN, SPL, and SPP under salt stress. This locus may be useful in the breeding program of marker-assisted selection for improving salt tolerance of barley. However, QTLs associated with NA, K, and NAK differed greatly between non-stress and salt stress environments. It may be suggested that only the QTLs detected under salt stress are really associated with salt tolerance in barley.

Draft genome sequence, and a sequence-defined genetic linkage map of the legume crop species Lupinus angustifolius L.

Lupin (Lupinus angustifolius L.) is the most recently domesticated crop in major agricultural cultivation. Its seeds are high in protein and dietary fibre, but low in oil and starch. Medical and dietetic studies have shown that consuming lupin-enriched food has significant health benefits. We report the draft assembly from a whole genome shotgun sequencing dataset for this legume species with 26.9x coverage of the genome, which is predicted to contain 57,807 genes. Analysis of the annotated genes with metabolic pathways provided a partial understanding of some key features of lupin, such as the amino acid profile of storage proteins in seeds. Furthermore, we applied the NGS-based RAD-sequencing technology to obtain 8,244 sequence-defined markers for anchoring the genomic sequences. A total of 4,214 scaffolds from the genome sequence assembly were aligned into the genetic map. The combination of the draft assembly and a sequence-defined genetic map made it possible to locate and study functional genes of agronomic interest. The identification of co-segregating SNP markers, scaffold sequences and gene annotation facilitated the identification of a candidate R gene associated with resistance to the major lupin disease anthracnose. We demonstrated that the combination of medium-depth genome sequencing and a high-density genetic linkage map by application of NGS technology is a cost-effective approach to generating genome sequence data and a large number of molecular markers to study the genomics, genetics and functional genes of lupin, and to apply them to molecular plant breeding. This strategy does not require prior genome knowledge, which potentiates its application to a wide range of non-model species.