The density and utility of the molecular genetic linkage map of the widespread use of RFLP markers and maps in suncultivated sunflower (Helianthus annuus L.) has been greatly inflower has been restricted by a lack of public RFLP creased by the development and mapping of several hundred simple sequence repeat (SSR) markers. Of 1089 public SSR markers de-probes, consequent lack of a dense public RFLP map, scribed thus far, 408 have been mapped in a recombinant inbred line and low-throughput nature of RFLP markers. The diffi-(RIL) mapping population (RHA280 ϫ RHA801). The goal of the culties posed by the historic lack of public, single-copy present research was to increase the density of the sunflower map by DNA markers were only weakly offset by the emerconstructing a new RIL map (PHA ϫ PHB) based on SSRs, adding gence of facile, universal DNA markers, e.g., RAPDs loci for newly developed SSR markers to the RHA280 ϫ RHA801 RIL (Williams et al., 1990, 1993) and AFLPs (Vos et al., map, and integrating the restriction fragment length polymorphism 1995). RAPDs have primarily been used for tagging (RFLP) and SSR maps of sunflower. The latter was accomplished by phenotypic loci in sunflower, e.g., rust (Puccinia helianadding 120 SSR marker loci to a backbone of 80 RFLP marker loci thi Schw.) and Orobanche cumana Wallr. resistance on the HA370 ϫ HA372 F 2 map. The map spanned 1275.4 centimorgans (cM) and had a mean density of 6.3 cM per locus. The genes (Lawson et al., 1998; Lu et al., 2000). While RAPD PHA ϫ PHB SSR map was constructed from 264 SSR marker loci, and AFLP markers have a multitude of uses, both are spanned 1199.4 cM, and had a mean density of 4.5 cM per locus. The dominant, multicopy, and often nonspecific in nature RHA280 ϫ RHA801 map was constructed by adding 118 new SSR and, as a whole, unsatisfactory for establishing a geand insertion-deletion (INDEL) marker loci to 459 previously nome-wide framework of DNA markers for anchoring mapped SSR marker loci. The 577-locus map spanned 1423.0 cM and cross referencing genetic linkage maps. Single-copy, and had a mean density of 2.5 cM per locus. The three maps were codominant DNA markers, e.g., SSRs, are preferred for constructed from 1044 DNA marker loci (701 unique SSR and 89 such purposes and, until recently, have been lacking unique RFLP or INDEL marker loci) and supply a dense genomein sunflower. wide framework of sequence-based DNA markers for molecular breeding and genomics research in sunflower.

A mapping population was created to detect quantitative trait loci (QTL) for resistance to stem rust caused by Puccinia graminis subsp. graminicola in Lolium perenne. A susceptible and a resistant plant were crossed to produce a pseudo-testcross population of 193 F(1) individuals. Markers were produced by the restriction-site associated DNA (RAD) process, which uses massively parallel and multiplexed sequencing of reduced-representation libraries. Additional simple sequence repeat (SSR) and sequence-tagged site (STS) markers were combined with the RAD markers to produce maps for the female (738 cM) and male (721 cM) parents. Stem rust phenotypes (number of pustules per plant) were determined in replicated greenhouse trials by inoculation with a field-collected, genetically heterogeneous population of urediniospores. The F(1) progeny displayed continuous distribution of phenotypes and transgressive segregation. We detected three resistance QTL. The most prominent QTL (qLpPg1) is located near 41 cM on linkage group (LG) 7 with a 2-LOD interval of 8 cM, and accounts for 30-38% of the stem rust phenotypic variance. QTL were detected also on LG1 (qLpPg2) and LG6 (qLpPg3), each accounting for approximately 10% of phenotypic variance. Alleles of loci closely linked to these QTL originated from the resistant parent for qLpPg1 and from both parents for qLpPg2 and qLpPg3. Observed quantitative nature of the resistance may be due to partial-resistance effects against all pathogen genotypes, or qualitative effects completely preventing infection by only some genotypes in the genetically mixed inoculum. RAD markers facilitated rapid construction of new genetic maps in this outcrossing species and will enable development of sequence-based markers linked to stem rust resistance in L. perenne.

Wild biotypes of cultivated sunflower ( Helianthus annuus L.) are weeds in corn ( Zea mays L.), soybean ( Glycine max L.), and other crops in North America, and are commonly controlled by applying acetohydroxyacid synthase (AHAS)-inhibiting herbicides. Biotypes resistant to two classes of AHAS-inhibiting herbicides-imidazolinones (IMIs) or sulfonylureas (SUs)-have been discovered in wild sunflower populations (ANN-PUR and ANN-KAN) treated with imazethapyr or chlorsulfuron, respectively. The goals of the present study were to isolate AHAS genes from sunflower, identify mutations in AHAS genes conferring herbicide resistance in ANN-PUR and ANN-KAN, and develop tools for marker-assisted selection (MAS) of herbicide resistance genes in sunflower. Three AHAS genes ( AHAS1, AHAS2, and AHAS3) were identified, cloned, and sequenced from herbicide-resistant (mutant) and -susceptible (wild type) genotypes. We identified 48 single-nucleotide polymorphisms (SNPs) in AHAS1, a single six-base pair insertion-deletion in AHAS2, and a single SNP in AHAS3. No DNA polymorphisms were found in AHAS2 among elite inbred lines. AHAS1 from imazethapyr-resistant inbreds harbored a C-to-T mutation in codon 205 ( Arabidopsis thaliana codon nomenclature), conferring resistance to IMI herbicides, whereas AHAS1 from chlorsulfuron-resistant inbreds harbored a C-to-T mutation in codon 197, conferring resistance to SU herbicides. SNP and single-strand conformational polymorphism markers for AHAS1, AHAS2, and AHAS3 were developed and genetically mapped. AHAS1, AHAS2, and AHAS3 mapped to linkage groups 2 ( AHAS3), 6 ( AHAS2), and 9 ( AHAS1). The C/T SNP in codon 205 of AHAS1 cosegregated with a partially dominant gene for resistance to IMI herbicides in two mutant x wild-type populations. The molecular breeding tools described herein create the basis for rapidly identifying new mutations in AHAS and performing MAS for herbicide resistance genes in sunflower.

Simple sequence repeat (SSR) markers were developed for cultivated sunflower (Helianthus annuus L.) from the DNA sequences of 970 clones isolated from genomic DNA libraries enriched for (CA)n,, (CT)n, (CAA)n, (CATA)n, or (GATA)n. The clones harbored 632 SSRs, of which 259 were unique. SSR markers were developed for 130 unique SSRs by designing and testing primers for 171 unique SSRs. Of the total, 74 SSR markers were polymorphic when screened for length polymorphisms among 16 elite inbred lines. The mean number of alleles per locus was 3.7 for dinucleotide, 3.6 for trinucleotide, and 9.5 for tetranucleotide repeats and the mean polymorphic information content (PIC) scores were 0.53 for dinucleotide, 0.53 for trinucleotide, and 0.83 for tetranucleotide repeats. Cluster analyses uncovered patterns of genetic diversity concordant with patterns produced by RFLP fingerprinting. SSRs were found to be slightly more polymorphic than RFLPs. Several individual SSRs were significantly more polymorphic than RFLP and other DNA markers in sunflower (20% of the polymorphic SSR markers had PIC scores ranging from 0.70 to 0.93). The newly developed SSRs greatly increase the supply of sequence-based DNA markers for DNA fingerprinting, genetic mapping, and molecular breeding in sunflower; however, several hundred additional SSR markers are needed to routinely construct complete genetic maps and saturate the genome.

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