Genetic structure and ecological niche space of lentil's closest wild relative, Lens orientalis (Boiss.) Schmalh.

Crops arose from wild ancestors and to understand their domestication it is essential to compare the cultivated species with their crop wild relatives. These represent an important source of further crop improvement, in particular in relation to climate change. Although there are about 58,000 Lens accessions held in genebanks, only 1% are wild. We examined the geographic distribution and genetic diversity of the lentil's immediate progenitor L. orientalis. We used Genotyping by Sequencing (GBS) to identify and characterize differentiation among accessions held at germplasm collections. We then determined whether genetically distinct clusters of accessions had been collected from climatically distinct locations. Of the 195 genotyped accessions, 124 were genuine L. orientalis with four identified genetic groups. Although an environmental distance matrix was significantly correlated with geographic distance in a Mantel test, the four identified genetic clusters were not found to occupy significantly different environmental space. Maxent modelling gave a distinct predicted distribution pattern centred in the Fertile Crescent, with intermediate probabilities of occurrence in parts of Turkey, Greece, Cyprus, Morocco, and the south of the Iberian Peninsula with NW Africa. Future projections did not show any dramatic alterations in the distribution according to the climate change scenarios tested. We have found considerable diversity in L. orientalis, some of which track climatic variability. The results of the study showed the genetic diversity of wild lentil and indicate the importance of ongoing collections and in situ conservation for our future capacity to harness the genetic variation of the lentil progenitor.

A hypervariable intron of the STAYGREEN locus provides excellent discrimination among Pisum fulvum accessions and reveals evidence for a relatively recent hybridization event with Pisum sativum.

An analysis of 82 non-synonymous Pisum fulvum accessions for sequence variation in a fragment of the STAYGREEN (SGR) locus revealed 57 alleles, most of which differed in indel structure. Eight additional P. fulvum accessions, each supposedly synonymous with a different accession of the initial group, were also analyzed. In every case the paired synonymous accessions possessed the same SGR sequence but varied slightly for a 6-trait morphological phenotype, indicating that SGR sequence is a much more reliable indicator of accession identity than is a morphological characterization. SGR sequence analysis confirmed our previous finding that P. fulvum accessions separate into two allele groups. This division was not supported by results of previous studies that were based on sequences distributed across the entire genome, suggesting that the division may have been produced by selection at a nearby locus and that the SGR phylogeny may not be good indicator of overall relationships within the species. One P. fulvum accession, PI 595941 (=JI1796), displayed an SGR sequence outside the variation typical of the species. Instead, its allele resembled alleles limited to a set of Pisum sativum landraces from the Middle East, suggesting hybridization between ancestors of PI 595941 and some primitive form of domesticated P. sativum. With one exception from the extreme northwest corner of Israel, P. fulvum accessions collected north of latitude 35.5° N were fixed for alleles from group A. These northern accessions also displayed greatly reduced SGR sequence diversity compared to group A accessions collected from other regions, suggesting that the northern populations may represent recent extensions of the range of the species. Group B accessions were distributed from Lake Tiberias south and were generally sympatric with the southern group A accessions. Although group B accessions occupied a smaller area than group A, the SGR sequence diversity in this group (28 alleles in 33 accessions) exceeded that for group A.

First Report of Bean yellow mosaic virus from Diseased Lupinus luteus in Eastern Washington.

Lupine accessions from the Cool Season Food Legume Seed Collection are grown for seed regenerations in Pullman, WA by the Agricultural Research Service, Western Regional Plant Introduction Station. Selected seed was germinated in the greenhouse and assayed by indirect ELISA using antiserum for potyvirus group detection (Agdia, Inc., Elkhart, IN). Healthy transplants were grown for seed collection on outside plots. In July of 2005, more than 90% of 307 Lupinus luteus L. transplants developed severe yellowing, necrosis, and stunting with an estimated 5% plant death. Plants were heavily infested with aphids and leaf sap was serologically positive for potyvirus. Partially purified virus preparations from infected plants contained filamentous particles and a 35-kDa protein that reacted with universal potyvirus antiserum on western blots. Reverse transcription (RT)-PCR using potyvirus universal primers (2) and cDNA derived from virion RNA generated a ~1.7-kbp product that was cloned and sequenced. The sequenced portion of the genomic RNA contained 1,610 nucleotides (nt) on its 3'-terminus (GenBank Accession No. EU144223) that included a partial nuclear inclusion protein, NIb, (1 to 637 nt) with the conserved amino acid (aa) replicase motif GDD (131 to 139 nt), the coat protein (CP) gene of 821 nt (638 to 1,459 nt), and a 171-nt untranslated region (1,460 to 1,630 nt) attached to a poly(A)tail. The CP sequence contained a NAG motif instead of the DAG motif commonly associated with aphid transmission. Searches in the NCBI GenBank database revealed that the CP aa and nt sequences contained conserved domains with isolates of Bean yellow mosaic virus (BYMV). A pairwise alignment (ClustalX) (4) of the CP aa from 20 BYMV isolates with the BYMV-Pullman isolate revealed identities from 96% (BYMV-S, U47033) to 88% (BYMV-MI [X81124)] -MI-NAT [AF434661]). This meets the species demarcation criteria of more than ~80% identity for inclusion with BYMV (1). Virion mechanical inoculations resulted in local lesions on Chenopodium amaranticolor Coste et Reyn and C. quinoa Willd., necrotic blotches on Phaseolus vulgaris L., and yellow spots and systemic movement in L. succulentus Douglas ex. K. Koch, L. texensis 'Bluebonnet', and L. texensis 'Maroon'; BYMV was confirmed by western blots and ELISA. The experimental inoculations represent the first documented report of BYMV in the annual L. succulentus and biennial L. texensis species. Since BYMV is seedborne and transmitted by many aphid species (3), it is possible that several lupine transplants escaped potyvirus detection, and secondary transmission of BYMV to plants occurred by aphids. During the 1950s, BYMV was confirmed in several annual lupines grown as crops in the southeastern United States (3). To our knowledge, this is the first report of BYMV occurring naturally in a lupine species in Washington. BYMV is a destructive virus to lupine species worldwide and has a wide host range in Fabaceae. This research directly contributes toward the maintenance of virus-free lupine seed for distribution to scientists focusing on lupine research. References: (1) P. H. Berger et al. Family Potyviridae. Page 819 in: Virus Taxonomy: Eighth Report of the ICTV. C. M. Fauquet et al. eds., 2005. (2) J. Chen et al. Arch. Virol. 146:757, 2001. (3) R. A. C. Jones and G. D. Mclean, Ann. Appl. Biol. 114:609, 1989. (4) J. D. Thompson et al. Nucleic Acids Res. 24:4878, 1997.

Construction and characterization of two bacterial artificial chromosome libraries of pea (Pisum sativum L.) for the isolation of economically important genes.

Pea (Pisum sativum L.) has a genome of about 4 Gb that appears to share conserved synteny with model legumes having genomes of 0.2-0.4 Gb despite extensive intergenic expansion. Pea plant inventory (PI) accession 269818 has been used to introgress genetic diversity into the cultivated germplasm pool. The aim here was to develop pea bacterial artificial chromosome (BAC) libraries that would enable the isolation of genes involved in plant disease resistance or control of economically important traits. The BAC libraries encompassed about 3.2 haploid genome equivalents consisting of partially HindIII-digested DNA fragments with a mean size of 105 kb that were inserted in 1 of 2 vectors. The low-copy oriT-based T-DNA vector (pCLD04541) library contained 55 680 clones. The single-copy oriS-based vector (pIndigoBAC-5) library contained 65 280 clones. Colony hybridization of a universal chloroplast probe indicated that about 1% of clones in the libraries were of chloroplast origin. The presence of about 0.1% empty vectors was inferred by white/blue colony plate counts. The usefulness of the libraries was tested by 2 replicated methods. First, high-density filters were probed with low copy number sequences. Second, BAC plate-pool DNA was used successfully to PCR amplify 7 of 9 published pea resistance gene analogs (RGAs) and several other low copy number pea sequences. Individual BAC clones encoding specific sequences were identified. Therefore, the HindIII BAC libraries of pea, based on germplasm accession PI 269818, will be useful for the isolation of genes underlying disease resistance and other economically important traits.

Consistent Quantitative Trait Loci in Pea for Partial Resistance to Aphanomyces euteiches Isolates from the United States and France.

ABSTRACT Development of pea cultivars resistant to Aphanomyces root rot, the most destructive root disease of pea worldwide, is a major disease management objective. In a previous study of a mapping population of 127 recombinant inbred lines (RILs) derived from the cross 'Puget' (susceptible) x '90-2079' (partially resistant), we identified seven genomic regions, including a major quantitative trait locus (QTL), Aph1, associated with partial resistance to Aphanomyces root rot in U.S. fields (21). The objective of the present study was to evaluate, in the same mapping population, the specificity versus consistency of Aphanomyces resistance QTL under two screening conditions (greenhouse and field, by comparison with the previous study) and with two isolates of Aphanomyces euteiches originating from the United States and France. The 127 RILs were evaluated in the greenhouse for resistance to pure culture isolates SP7 (United States) and Ae106 (France). Using the genetic map previously described, a total of 10 QTL were identified for resistance in greenhouse conditions to the two isolates. Among these were Aph1, Aph2, and Aph3, previously detected for partial field resistance in the United States. Aph1 and Aph3 were detected with both isolates and Aph2 with only the French isolate. Seven additional QTL were specifically detected with one of the two isolates and were not identified for partial field resistance in the United States. The consistency of the detected resistance QTL over two screening environments and isolates is discussed with regard to pathogen variability, and disease assessment and QTL detection methods. This study suggests the usefulness of three consistent QTL, Aph1, Aph2, and Aph3, for marker-assisted selection.

Quantitative trait loci for partial resistance to Aphanomyces root rot in pea.

Aphanomyces root rot, caused by Aphanomyces euteiches Drechs, is the most-important disease of pea ( Pisum sativum L.) worldwide. No efficient chemicals are available to control the pathogen. To facilitate breeding for Aphanomyces root rot resistance and to better understand the inheritance of partial resistance, our goal was to identify QTLs associated with field partial resistance. A population of 127 RILs from the cross Puget (susceptible) x 90-2079 (partially resistant) was used. The lines were assessed for resistance to A. euteiches under field conditions at two locations in the United States (Pullman, Wash. and LeSueur, Minn.) in 1996 and 1998 for three criteria based on symptom intensity and disease effects on the whole plant. The RILs were genotyped using automated AFLPs, RAPDs, SSRs, ISSRs, STSs, isozymes and morphological markers. The resulting genetic map consisted of 324 linked markers distributed over 13 linkage groups covering 1,094 cM (Kosambi). Twenty seven markers were anchored to other published pea genetic maps. A total of seven genomic regions were associated with Aphanomyces root rot resistance. The first one, located on LG IVb and named Aph1, was considered as "major" since it was highly consistent over the years, locations and resistance criteria studied, and it explained up to 47% of the variation in the 1998 Minnesota trial. Two other year-specific QTLs, namely Aph2 and Aph3, were revealed from different scoring criteria on LG V and Ia, respectively. Aph2 and Aph3 mapped near the r (wrinkled/round seeds) and af (normal/afila leaves) genes, and accounted for up to 32% and 11% of the variation, respectively. Four other "minor" QTLs, identified on LG Ib, VII and B, were specific to one environment and one resistance criterion. The resistance alleles of Aph3 and the two "minor" QTLs on LG Ib were derived from the susceptible parent. Flanking markers for the major Aphanomyces resistance QTL, Aph1, have been identified for use in marker-assisted selection to improve breeding efficiency.