Mark D. Burow

Comparative genomics of plant chromosomes

Comparative genomics, the study of similarities and differences in structure and function of the hereditary information in different taxa, uses molecular tools to investigate notions that far preceded the discovery that DNA was the hereditary molecule. Vavilov’s (1922) “law of homologous series in variation” was an early suggestion of the possibility of underlying commonality in the genetic blueprints of different (plant) species. In plants, genetic analysis based upon morphological and isozyme markers provided early hints that the arrangements of genes along the chromosomes of various taxa may have retained parallels since their divergence from common ancestors. DNA-level investigations in diverse taxa point to two broad messages: (1) The small but essential portion of most plant genomes encoding genes evolves relatively slowly, with corresponding genes retaining recognizable DNA sequences and similar order along the chromosomes of taxa that have been reproductively-isolated for millions of years. (2) A wide range of factors, such as DNA sequence mobility, gene deletion, and localized rearrangements, are superimposed on the relatively slow tempo of chromosomal evolution and cause many deviations from co-linearity. (3) Genetic loci that account for common phenotypes in different taxa are often at corresponding genomic locations, and may represent orthologous genes or members of orthologous clusters of genes.

Microsatellite identification and characterization in peanut (A. hypogaea L.)

A major constraint to the application of biotechnology to the improvement of the allotetraploid peanut, or groundnut (Arachis hypogaea L.), has been the paucity of polymorphism among germplasm lines using biochemical (seed proteins, isozymes) and DNA markers (RFLPs and RAPDs). Six sequence-tagged microsatellite (STMS) markers were previously available that revealed polymorphism in cultivated peanut. Here, we identify and characterize 110 STMS markers that reveal genetic variation in a diverse array of 24 peanut landraces. The simple-sequence repeats (SSRs) were identified with a probe of two 27,648-clone genomic libraries: one constructed using PstI and the other using Sau3AI/BamHI. The most frequent, repeat motifs identified were ATT and GA, which represented 29% and 28%, respectively, of all SSRs identified. These were followed by AT, CTT, and GT. Of the amplifiable primers, 81% of ATT and 70.8% of GA repeats were polymorphic in the cultivated peanut test array. The repeat motif AT showed the maximum number of alleles per locus (5.7). Motifs ATT, GT, and GA had a mean number of alleles per locus of 4.8, 3.8, and 3.6, respectively. The high mean number of alleles per polymorphic locus, combined with their relative frequency in the genome and amenability to probing, make ATT and GA the most useful and appropriate motifs to target to generate further SSR markers for peanut.

The genome sequences of Arachis duranensis and Arachis ipaensis , the diploid ancestors of cultivated peanut

Cultivated peanut (Arachis hypogaea) is an allotetraploid with closely related subgenomes of a total size of ∼2.7 Gb. This makes the assembly of chromosomal pseudomolecules very challenging. As a foundation to understanding the genome of cultivated peanut, we report the genome sequences of its diploid ancestors (Arachis duranensis and Arachis ipaensis). We show that these genomes are similar to cultivated peanuts A and B subgenomes and use them to identify candidate disease resistance genes, to guide tetraploid transcript assemblies and to detect genetic exchange between cultivated peanuts subgenomes. On the basis of remarkably high DNA identity of the A. ipaensis genome and the B subgenome of cultivated peanut and biogeographic evidence, we conclude that A. ipaensis may be a direct descendant of the same population that contributed the B subgenome to cultivated peanut.

Physiology and proteomics of the water-deficit stress response in three contrasting peanut genotypes.

Peanut genotypes from the US mini-core collection were analysed for changes in leaf proteins during reproductive stage growth under water-deficit stress. One- and two-dimensional gel electrophoresis (1- and 2-DGE) was performed on soluble protein extracts of selected tolerant and susceptible genotypes. A total of 102 protein bands/spots were analysed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and by quadrupole time-of-flight tandem mass spectrometry (Q-TOF MS/MS) analysis. Forty-nine non-redundant proteins were identified, implicating a variety of stress response mechanisms in peanut. Lipoxygenase and 1l-myo-inositol-1-phosphate synthase, which aid in inter- and intracellular stress signalling, were more abundant in tolerant genotypes under water-deficit stress. Acetyl-CoA carboxylase, a key enzyme of lipid biosynthesis, increased in relative abundance along with a corresponding increase in epicuticular wax content in the tolerant genotype, suggesting an additional mechanism for water conservation and stress tolerance. Additionally, there was a marked decrease in the abundance of several photosynthetic proteins in the tolerant genotype, along with a concomitant decrease in net photosynthesis in response to water-deficit stress. Differential regulation of leaf proteins involved in a variety of cellular functions (e.g. cell wall strengthening, signal transduction, energy metabolism, cellular detoxification and gene regulation) indicates that these molecules could affect the molecular mechanism of water-deficit stress tolerance in peanut.

Molecular mapping of genes conferring aluminum tolerance in rice (Oryza sativa L.)

Abstract Crop productivity on acid soil is restricted by multiple abiotic stress factors. Aluminum (Al) tolerance seems to be a key to productivity on soil with a pH below 5.0, but other factors such as Mn toxicity and the deficiency of P, Ca and Mg also play a role. The development of Al-tolerant genotypes of rice is an urgent necessity for improving crop productivity in developing countries. Inhibition of root growth is a primary and early symptom of Al toxicity. The present study was conducted to identify genetic factors controlling the aluminum tolerance of rice. Several parameters related to Al tolerance, most importantly the relative root growth under Al stress versus non-stress conditions, were scored in 188 F3 selfed families from a cross between an Al-tolerant Vietnamese local variety, Chiembau, and an Al-susceptible improved variety, Omon269–65. The two varieties are both Oryza sativa ssp. indica, but showed a relatively high level of DNA polymorphism, permitting the assembly of an RFLP map consisting of 164 loci spanning 1,715.8 cM, and covering most of the rice genome. A total of nine different genomic regions on eight chromosomes have been implicated in the genetic control of root and shoot growth under aluminum stress. By far the greatest effects on aluminum tolerance were associated with the region near WG110 on chromosome 1. This region does not seem to correspond to most of the genes that have been mapped for aluminum tolerance in other species, nor do they correspond closely to one another. Most results, both from physiological studies and from molecular mapping studies, tend to suggest that aluminum tolerance is a complex multi-genic trait. The identification of DNA markers (such as WG110) that are diagnostic for aluminum tolerance in particular gene pools provides an important starting point for transferring and pyramiding genes that may contribute to the sustainable improvement of crop productivity in aluminum-rich soils. The isolation of genes responsible for aluminum tolerance is likely to be necessary to gain a comprehensive understanding of this complex trait.

Identification of peanut (Arachis hypogaea L.) RAPD markers diagnostic of root-knot nematode (Meloidogyne arenaria (Neal) Chitwood) resistance

DNA markers linked to a root-knot nematode resistance gene derived from wild peanut species have been identified. The wild diploid peanut accessions K9484 (Arachis batizocoi Krapov. & W. C. Gregory), GKP10017, (A. cardenasii Krapov & W. C. Gregory), and GKP10602 (A. diogoi Hoehne) possess genes for ressitance to Meloidogyne arenaria. These three accessions and A. hypogaea cv. Florunner were crossed to generate the hybrid resistant breeding line TxAg-7. This line was used as donor parent to develop a BC4F2 population segregating for resistance. Three RAPD markers associated with nematode resistance were identified in this population by bulked segregant analysis. Linkage was confirmed by screening 21 segregatingh BC4F2 and 63 BC5F2 single plants. Recombination between marker RKN410 and resistance, and between marker RKN440 and resistance, was estimated to be 5.4±1.9% and 5.8±2.1%, respectively, on a per-generation basis. These two markers identified a resistance gene derived from either A. cardenasii or A. diogoi, and were closely linked to each other. Recombination between a third marker, RKN229, inherited from A. cardenasii or A. diogoi, and resistance was 9.0±3.2% per generation. Markers RKN410 and RKN229 appeared to be linked genetically and flank the same resistance gene. All markers were confirmed by hybridization of cloned or gel-purified marker DNA to blots of PCR-amplified DNA. Pooled data on the segregation of BC5F2 plants was consistent with the presence of one resistance gene in the advanced breeding lines. Different distributions of resistance in the BC5F2 progeny and TxAG-7 suggest the presence of additional resistance genes in TxAG-7.

High-frequency generation of transgenic tobacco plants after modified leaf disk cocultivation withAgrobacterium tumefaciens

A modified protocol for theAgrobacterium tumefaciens-mediated transformation of tobacco (Nicotina tabacum L.) leaf disks was developed for greater recovery of transgenic plants. Modifications include transformation ofAgrobacterium by a freeze-thaw procedure, initial cocultivation of leaf disks andAgrobacterium under vacuum, subsequent growth with nurse cells for one week, rooting of shoots in medium lacking carbenicillin, longer, growth in rooting medium, and a shortened “hardening” step. By this procedure, an average of 1.3 kanamycin-resistant calli were obtained per leaf disk, and 38% of, the callus cultures used were regenerated to produce 133 independently transformed tobacco plants.

Isolation of cDNA clones of genes induced upon transfer of Chlamydomonas reinhardtii cells to low CO2

Unicellular algae grow well under limiting CO2 conditions, aided by a carbon concentrating mechanism (CCM). In C. reinhardtii, this mechanism is inducible and is present only in cells grown under low CO2 conditions. We constructed a cDNA library from cells adapting to low CO2, and screened the library for cDNAs specific to low CO2-adapting cells. Six classes of low CO2-inducible clones were identified. One class of clone, reported here, represents a novel gene associated with adaptation of cells to air. A second class of clones corresponds to the air-inducible periplasmic carbonic anhydrase I (CAH1). These clones represent genes that respond to the level of CO2 in the environment.

Gene expression profiling in peanut using high density oligonucleotide microarrays

BackgroundTranscriptome expression analysis in peanut to date has been limited to a relatively small set of genes and only recently has a significant number of ESTs been released into the public domain. Utilization of these ESTs for oligonucleotide microarrays provides a means to investigate large-scale transcript responses to a variety of developmental and environmental signals, ultimately improving our understanding of plant biology.ResultsWe have developed a high-density oligonucleotide microarray for peanut using 49,205 publicly available ESTs and tested the utility of this array for expression profiling in a variety of peanut tissues. To identify putatively tissue-specific genes and demonstrate the utility of this array for expression profiling in a variety of peanut tissues, we compared transcript levels in pod, peg, leaf, stem, and root tissues. Results from this experiment showed 108 putatively pod-specific/abundant genes, as well as transcripts whose expression was low or undetected in pod compared to peg, leaf, stem, or root. The transcripts significantly over-represented in pod include genes responsible for seed storage proteins and desiccation (e.g., late-embryogenesis abundant proteins, aquaporins, legumin B), oil production, and cellular defense. Additionally, almost half of the pod-abundant genes represent unknown genes allowing for the possibility of associating putative function to these previously uncharacterized genes.ConclusionThe peanut oligonucleotide array represents the majority of publicly available peanut ESTs and can be used as a tool for expression profiling studies in diverse tissues.

Identification of QTLs for pod and kernel traits in cultivated peanut by bulked segregant analysis

Bulked segregant analysis was used to identify simple sequence repeat (SSR) markers associated with pod and kernel traits in cultivated peanut, to permit rapid selection of superior quality genotypes in the breeding program. SSR markers linked to pod and kernel traits were identified in two DNA pools (high and low), which were established using selected F 2:6 recombinant individuals resulting from a cultivated cross between a runner (Tamrun OL01) and a Spanish (BSS 56) peanut. To identify quantitative trait loci (QTLs) for pod and kernel-related traits, parents were screened initially with 112 SSR primer pairs. The survey revealed 8.9% polymorphism between parents. Of ten SSR primer pairs distinguishing the parents, five (PM375, PM36, PM45, pPGPseq8D9, and Ah-041) were associated with differences between bulks for seed length, pod length, number of pods per plant, 100-seed weight, maturity, or oil content. Association was confirmed by analysis of segregation among 88 F 2:6 individuals in the RIL population. Phenotypic means associated with markers for three traits differed by more than 40%, indicating the presence of QTLs with major effects for number of pods per plant, plant weight, and pod maturity. The SSR markers can be used for marker assisted selection for quality and yield improvement in peanut. To the best of our knowledge, this is the first report on the identification of SSR markers linked to pod - and kernel- related traits in cultivated peanut.