Khalid Meksem

TILLING to detect induced mutations in soybean

BackgroundSoybean (Glycine max L. Merr.) is an important nitrogen-fixing crop that provides much of the worlds protein and oil. However, the available tools for investigation of soybean gene function are limited. Nevertheless, chemical mutagenesis can be applied to soybean followed by screening for mutations in a target of interest using a strategy known as Targeting Induced Local Lesions IN Genomes (TILLING). We have applied TILLING to four mutagenized soybean populations, three of which were treated with ethyl methanesulfonate (EMS) and one with N-nitroso-N-methylurea (NMU).ResultsWe screened seven targets in each population and discovered a total of 116 induced mutations. The NMU-treated population and one EMS mutagenized population had similar mutation density (~1/140 kb), while another EMS population had a mutation density of ~1/250 kb. The remaining population had a mutation density of ~1/550 kb. Because of soybeans polyploid history, PCR amplification of multiple targets could impede mutation discovery. Indeed, one set of primers tested in this study amplified more than a single target and produced low quality data. To address this problem, we removed an extraneous target by pretreating genomic DNA with a restriction enzyme. Digestion of the template eliminated amplification of the extraneous target and allowed the identification of four additional mutant alleles compared to untreated template.ConclusionThe development of four independent populations with considerable mutation density, together with an additional method for screening closely related targets, indicates that soybean is a suitable organism for high-throughput mutation discovery even with its extensively duplicated genome.

A soybean cyst nematode resistance gene points to a new mechanism of plant resistance to pathogens

Soybean (Glycine max (L.) Merr.) is an important crop that provides a sustainable source of protein and oil worldwide. Soybean cyst nematode (Heterodera glycines Ichinohe) is a microscopic roundworm that feeds on the roots of soybean and is a major constraint to soybean production. This nematode causes more than US

'Forrest' resistance to the soybean cyst nematode is bigenic : saturation mapping of the Rhg1 and Rhg4 loci

1 billion in yield losses annually in the United States alone, making it the most economically important pathogen on soybean. Although planting of resistant cultivars forms the core management strategy for this pathogen, nothing is known about the nature of resistance. Moreover, the increase in virulent populations of this parasite on most known resistance sources necessitates the development of novel approaches for control. Here we report the map-based cloning of a gene at the Rhg4 (for resistance to Heterodera glycines 4) locus, a major quantitative trait locus contributing to resistance to this pathogen. Mutation analysis, gene silencing and transgenic complementation confirm that the gene confers resistance. The gene encodes a serine hydroxymethyltransferase, an enzyme that is ubiquitous in nature and structurally conserved across kingdoms. The enzyme is responsible for interconversion of serine and glycine and is essential for cellular one-carbon metabolism. Alleles of Rhg4 conferring resistance or susceptibility differ by two genetic polymorphisms that alter a key regulatory property of the enzyme. Our discovery reveals an unprecedented plant resistance mechanism against a pathogen. The mechanistic knowledge of the resistance gene can be readily exploited to improve nematode resistance of soybean, an increasingly important global crop.

Microsatellite markers identify three additional quantitative trait loci for resistance to soybean sudden-death syndrome (SDS) in Essex × Forrest RILs

Abstract  Field resistance to cyst nematode (SCN) race 3 (Heterodera glycines I.) in soybean [Glycine max (L.) Merr.] cv ’Forrest’ is conditioned by two QTLs: the underlying genes are presumed to include Rhg1 on linkage group G and Rhg4 on linkage group A2. A population of recombinant inbred lines (RILs) and two populations of near-isogenic lines (NILs) derived from a cross of Forrest×Essex were used to map the loci affecting resistance to SCN. Bulked segregant analysis, with 512 AFLP primer combinations and microsatellite markers, produced a high-density genetic map for the intervals carrying Rhg1 and Rhg4. The two QTLs involved in resistance to SCN were strongly associated with the AFLP marker EATGMCGA87 (P=0.0001, R2=24.5%) on linkage group G, and the AFLP marker ECCGMAAC405 (P=0.0001, R2 =26.2%) on linkage group A2. Two- way analysis of variance showed epistasic interaction (P=0.0001, R2 =16%) between the two loci controlling SCN resistance in Essex×Forrest recombinant inbred lines. Considering the two loci as qualitative genes and the resistance as female index FI <5%, jointly the two loci explained over 98% of the resistance. The locations of the two QTLs were confirmed in the NILs populations. Therefore SCN resistance in Forrest×Essex is bigenic. High-efficiency marker-assisted selection can be performed using the markers to develop cultivars with stable resistance to SCN.

Common loci underlie field resistance to soybean sudden death syndrome in Forrest, Pyramid, Essex, and Douglas.

Abstract Resistance to the sudden-death syndrome (SDS) of soybean (Glycine max L. Merr.), caused by Fusarium solani f. sp. glycines, is controlled by a number of quantitatively inherited loci (QTLs). Forrest showed a strong field resistance to SDS while Essex is susceptible to SDS. A population of 100 recombinant inbred lines (RILs) derived from a cross of Essex × Forrest was used to map the loci effecting resistance to SDS using phenotypic data obtained from six environments. Six loci involved in resistance to SDS were identified in this population. Four of the QTLs identified by BARC-Satt214 (P = 0.0001, R2= 24.1%), BARC-Satt309 (P = 0.0001, R2 = 16.3), BARC-Satt570 (P = 0.0001, R2 = 19.2%) and a random amplified polymorphic DNA (RAPD) marker OEO21000 (P = 0.0031, R2=12.6) were located on linkage group (LG) G (Satt309 and OEO21000 were previously reported). Jointly the four QTLs on LG G explained 50% of the variation in SDS disease incidence (DI). All the QTLs on LG G derived the beneficial allele from Forrest. Two QTLs, BARC-Satt371 (P = 0.0019, R2 = 12%) on LG C2 (previously reported) and BARC-Satt354 (P = 0.0015, R2 = 11.5%) on LG I, derived their beneficial allele from Essex and jointly explained about 40% of the variation in SDS DI. Two-way and multi-way interactions indicated that gene action was additive among the loci underlying resistance to SDS. These results suggest that cultivars with durable resistance to SDS can be developed via gene pyramiding.

Clustering among loci underlying soybean resistance to Fusarium solani, SDS and SCN in near-isogenic lines

Soybean [Glycine max (L.) Merr.] sudden death syndrome (SDS) caused by Fusarium solani f. sp. glycines results in severe yield losses. Resistant cultivars offer the most-effective protection against yield losses but resistant cultivars such as ’Forrest’ and ’Pyramid’ vary in the nature of their response to SDS. Loci underlying SDS resistance in ’Essex’ × Forrest are well defined. Our objectives were to identify and characterize loci and alleles that underlie field resistance to SDS in Pyramid×’Douglas’. SDS disease incidence and disease severity were determined in replicated field trials in six environments over 4 years. One hundred and twelve polymorphic DNA markers were compared with SDS disease response among 90 recombinant inbred lines from the cross Pyramid×Douglas. Two quantitative trait loci (QTLs) for resistance to SDS derived their beneficial alleles from Pyramid, identified on linkage group G by BARC-Satt163 (261-bp allele, P=0.0005, R2=16.0%) and linkage group N by BARC-Satt080 (230-bp allele, P=0.0009, R2=15.6%). Beneficial alleles of both QTLs were previously identified in Forrest. A QTL for re- sistance to SDS on linkage group C2 identified by BARC-Satt307 (292-bp allele, P=0.0008, R2=13.6%) derived the beneficial allele from Douglas. A beneficial allele of this QTL was previously identified in Essex. Recombinant inbred lines that carry the beneficial alleles for all three QTLs for resistance to SDS were significantly (P≤0.05) more resistant than other recombinant inbred lines . Among these recombinant inbred lines resistance to SDS was environmentally stable. Therefore, gene pyramiding will be an effective method for developing cultivars with stable resistance to SDS.

The Soybean Genome Database (SoyGD): a browser for display of duplicated, polyploid, regions and sequence tagged sites on the integrated physical and genetic maps of Glycine max

Abstract In the soybean [Glycine max (L.) Merr.] cultivar ’Forrest’ a single chromosomal region underlies co-inheritance of field resistance of the sudden-death syndrome (SDS), caused by the fungus Fusarium solani (Mart.) Sacc. f. sp. glycines (Burk.) Snyd. & Hans. and soybean cyst nematode (SCN) race 3 (caused by Heterodera glycines Ichinohe). Our objectives were to verify that co-inheritance was derived from a single chromosomal region in near-isogenic lines and to separate component gene clusters. DNA markers were compared with a SDS leaf-scorch index (DX), F. solani root-infection severity (IS) and a SCN index of parasitism (IP) among 80 near-isogenic lines (NILs). The genomic region identified by the RFLP marker Bng122D was strongly associated (0.0004 ≤P≤ 0.006) with mean SDS DX (R2 > 16–38%) and IS (R2 > 38–73%), but only marginally associated with resistance to SCN. However, the linked (4.3–7.4 cM) microsatellite marker SATT309 was strongly associated with both resistance to SCN (0.0001 ≤P≤ 0.0003; R2 > 24–97%) and mean leaf DX (0.0001 ≤P≤ 0.0003; R2 > 25–63%), but not root IS. Recombination events among markers and traits enabled separation of the qualitative loci underlying resistance to SDS and SCN. Our data showed that resistance to SDS DX, SDS IS and SCN IP in Forrest may be caused by four genes in a cluster with two pairs in close linkage or by a two-gene cluster with each gene displaying pleiotropy, one conditioning SDS IS and DX and the other SCN IP and SDS DX.

Genetic and physical localization of the soybean Rpg1-b disease resistance gene reveals a complex locus containing several tightly linked families of NBS-LRR genes

Genomes that have been highly conserved following increases in ploidy (by duplication or hybridization) like Glycine max (soybean) present challenges during genome analysis. At the Soybean Genome Database (SoyGD) genome browser has, since 2002, integrated and served the publicly available soybean physical map, bacterial artificial chromosome (BAC) fingerprint database and genetic map associated genomic data. The browser shows both build 3 and build 4 contiguous sets of clones (contigs) of the soybean physical map. Build 4 consisted of 2854 contigs that encompassed 1.05 Gb and 404 high-quality DNA markers that anchored 742 contigs. Many DNA markers anchored sets of 2–8 different contigs. Each contig in the set represented a homologous region of related sequences. GBrowse was adapted to show sets of homologous contigs at all potential anchor points, spread laterally and prevented from overlapping. About 8064 minimum tiling path (MTP2) clones provided 13 473 BAC end sequences (BES) to decorate the physical map. Analyses of BES placed 2111 gene models, 40 marker anchors and 1053 new microsatellite markers on the map. Estimated sequence tag probes from 201 low-copy gene families located 613 paralogs. The genome browser portal showed each data type as a separate track. Tetraploid, octoploid, diploid and homologous regions are shown clearly in relation to an integrated genetic and physical map.

Definition of Soybean Genomic Regions That Control Seed Phytoestrogen Amounts

Alleles or tightly linked genes at the soybean (Glycine max L. Merr.) Rpg1 locus confer resistance to strains of Pseudomonas syringae pv. glycinea that express the avirulence genes avrB or avrRpm1. We have previously mapped Rpg1-b (the gene specific for avrB) to a cluster of resistance genes (R genes) with diverse specificities in molecular linkage group F. Here, we describe the high-resolution physical and genetic mapping of Rpg1-b to a 0.16-cM interval encompassed by two overlapping BAC clones spanning approximately 270 kilobases. Rpg1-b is part of a complex locus containing numerous genes related to previously characterized coiled coil-nucleotide binding site-leucine rich repeat (CC-NBS-LRR)-type R genes that are spread throughout this region. Phylogenetic and Southern blot analyses group these genes into four distinct subgroups, some of which are conserved in the common bean, Phaseolus vulgaris, indicating that this R gene cluster may predate the divergence of Phaseolus and Glycine. Members from different subgroups are physically intermixed and display a high level of polymorphism between soybean cultivars, suggesting that this region is rearranging at a high frequency. At least five CC-NBS-LRR-type genes cosegregate with Rpg1-b in our large mapping populations.

Comparative sequence analysis of Solanum and Arabidopsis in a hot spot for pathogen resistance on potato chromosome V reveals a patchwork of conserved and rapidly evolving genome segments

Soybean seeds contain large amounts of isoflavones or phytoestrogens such as genistein, daidzein, and glycitein that display biological effects when ingested by humans and animals. In seeds, the total amount, and amount of each type, of isoflavone varies by 5 fold between cultivars and locations. Isoflavone content and quality are one key to the biological effects of soy foods, dietary supplements, and nutraceuticals. Previously we had identified 6 loci (QTL) controlling isoflavone content using 150 DNA markers. This study aimed to identify and delimit loci underlying heritable variation in isoflavone content with additional DNA markers. We used a recombinant inbred line (RIL) population (n=100) derived from the cross of “Essex” by “Forrest,” two cultivars that contrast for isoflavone content. Seed isoflavone content of each RIL was determined by HPLC and compared against 240 polymorphic microsatellite markers by one-way analysis of variance. Two QTL that underlie seed isoflavone content were newly discovered. The additional markers confirmed and refined the positions of the six QTL already reported. The first new region anchored by the marker BARC_Satt063 was significantly associated with genistein (P=0.009, R2=29.5%) and daidzein (P=0.007 , R2=17.0%). The region is located on linkage group B2 and derived the beneficial allele from Essex. The second new region defined by the marker BARC_Satt129 was significantly associated with total glycitein (P=0.0005 , R2=32.0%). The region is located on linkage group D1a+Q and also derived the beneficial allele from Essex. Jointly the eight loci can explain the heritable variation in isoflavone content. The loci may be used to stabilize seed isoflavone content by selection and to isolate the underlying genes.