Marker-assisted pyramiding of QTLs for heat tolerance and escape upgrades heat resilience in rice (Oryza sativa L.).

KEY MESSAGE: This study demonstrated that pyramiding of early morning flowering and heat tolerance QTLs (qEMF3 and qHTSF4.1) in rice is an efficient approach to maintain high spikelet fertility under high-temperature stress at flowering stage. High temperature at flowering stage of rice causes low spikelet fertility and low yield. To cope with high-temperature stress brought by climate change, two strategies were proposed to develop heat-resilient rice varieties. One is to escape the high temperature by flowering early in the morning, another is to enhance tolerance to high-temperature stress per se. Two promising QTLs for early morning flowering (qEMF3) and heat tolerance (qHTSF4.1) were introgressed into IR64 background, and Near isogenic lines (NILs) IR64 + qEMF3 (IR64EMF3) and IR64 + qHTSF4.1 (IR64HT4) were developed in previous studies. In this study, a QTL pyramiding line IR64 + qHTSF4.1 + qEMF3 (IR64HT4EMF3) was developed by marker-assisted selection of the progenies of previous NILs. The NILs were subjected to different high-temperature regimes in the indoor growth chambers and different locations in the field. In the indoor growth chambers, when high temperature starts early (before 11:00 am), IR64HT4 and IR64HT4EMF3 had higher spikelet fertility than IR64EMF3; when high temperature comes later (after 11:00 am), IR64EMF3 and IR64HT4EMF3 had higher spikelet fertility than IR64HT4. The flowering pattern of the IR64HT4EMF3 was earlier than IR64HT4, but similar to IR64EMF3 in the glasshouse, field and indoor growth chambers. IR64HT4EMF3 showed higher spikelet fertility than IR64EMF3 and IR64HT4 in the field in the Philippines. Thus, combination of early morning flowering and heat tolerance QTLs is an elegant breeding strategy to cope with future extreme climate.

Comparative mapping in intraspecific populations uncovers a high degree of macrosynteny between A- and B-genome diploid species of peanut.

BACKGROUND: Cultivated peanut or groundnut (Arachis hypogaea L.) is an important oilseed crop with an allotetraploid genome (AABB, 2n = 4x = 40). Both the low level of genetic variation within the cultivated gene pool and its polyploid nature limit the utilization of molecular markers to explore genome structure and facilitate genetic improvement. Nevertheless, a wealth of genetic diversity exists in diploid Arachis species (2n = 2x = 20), which represent a valuable gene pool for cultivated peanut improvement. Interspecific populations have been used widely for genetic mapping in diploid species of Arachis. However, an intraspecific mapping strategy was essential to detect chromosomal rearrangements among species that could be obscured by mapping in interspecific populations. To develop intraspecific reference linkage maps and gain insights into karyotypic evolution within the genus, we comparatively mapped the A- and B-genome diploid species using intraspecific F2 populations. Exploring genome organization among diploid peanut species by comparative mapping will enhance our understanding of the cultivated tetraploid peanut genome. Moreover, new sources of molecular markers that are highly transferable between species and developed from expressed genes will be required to construct saturated genetic maps for peanut. RESULTS: A total of 2,138 EST-SSR (expressed sequence tag-simple sequence repeat) markers were developed by mining a tetraploid peanut EST assembly including 101,132 unigenes (37,916 contigs and 63,216 singletons) derived from 70,771 long-read (Sanger) and 270,957 short-read (454) sequences. A set of 97 SSR markers were also developed by mining 9,517 genomic survey sequences of Arachis. An SSR-based intraspecific linkage map was constructed using an F2 population derived from a cross between K 9484 (PI 298639) and GKBSPSc 30081 (PI 468327) in the B-genome species A. batizocoi. A high degree of macrosynteny was observed when comparing the homoeologous linkage groups between A (A. duranensis) and B (A. batizocoi) genomes. Comparison of the A- and B-genome genetic linkage maps also showed a total of five inversions and one major reciprocal translocation between two pairs of chromosomes under our current mapping resolution. CONCLUSIONS: Our findings will contribute to understanding tetraploid peanut genome origin and evolution and eventually promote its genetic improvement. The newly developed EST-SSR markers will enrich current molecular marker resources in peanut.

A high-density genetic map of Arachis duranensis, a diploid ancestor of cultivated peanut.

BACKGROUND: Cultivated peanut (Arachis hypogaea) is an allotetraploid species whose ancestral genomes are most likely derived from the A-genome species, A. duranensis, and the B-genome species, A. ipaensis. The very recent (several millennia) evolutionary origin of A. hypogaea has imposed a bottleneck for allelic and phenotypic diversity within the cultigen. However, wild diploid relatives are a rich source of alleles that could be used for crop improvement and their simpler genomes can be more easily analyzed while providing insight into the structure of the allotetraploid peanut genome. The objective of this research was to establish a high-density genetic map of the diploid species A. duranensis based on de novo generated EST databases. Arachis duranensis was chosen for mapping because it is the A-genome progenitor of cultivated peanut and also in order to circumvent the confounding effects of gene duplication associated with allopolyploidy in A. hypogaea. RESULTS: More than one million expressed sequence tag (EST) sequences generated from normalized cDNA libraries of A. duranensis were assembled into 81,116 unique transcripts. Mining this dataset, 1236 EST-SNP markers were developed between two A. duranensis accessions, PI 475887 and Grif 15036. An additional 300 SNP markers also were developed from genomic sequences representing conserved legume orthologs. Of the 1536 SNP markers, 1054 were placed on a genetic map. In addition, 598 EST-SSR markers identified in A. hypogaea assemblies were included in the map along with 37 disease resistance gene candidate (RGC) and 35 other previously published markers. In total, 1724 markers spanning 1081.3 cM over 10 linkage groups were mapped. Gene sequences that provided mapped markers were annotated using similarity searches in three different databases, and gene ontology descriptions were determined using the Medicago Gene Atlas and TAIR databases. Synteny analysis between A. duranensis, Medicago and Glycine revealed significant stretches of conserved gene clusters spread across the peanut genome. A higher level of colinearity was detected between A. duranensis and Glycine than with Medicago. CONCLUSIONS: The first high-density, gene-based linkage map for A. duranensis was generated that can serve as a reference map for both wild and cultivated Arachis species. The markers developed here are valuable resources for the peanut, and more broadly, to the legume research community. The A-genome map will have utility for fine mapping in other peanut species and has already had application for mapping a nematode resistance gene that was introgressed into A. hypogaea from A. cardenasii.

The sunflower (Helianthus annuus L.) genome reflects a recent history of biased accumulation of transposable elements.

Aside from polyploidy, transposable elements are the major drivers of genome size increases in plants. Thus, understanding the diversity and evolutionary dynamics of transposable elements in sunflower (Helianthus annuus L.), especially given its large genome size (∼3.5 Gb) and the well-documented cases of amplification of certain transposons within the genus, is of considerable importance for understanding the evolutionary history of this emerging model species. By analyzing approximately 25% of the sunflower genome from random sequence reads and assembled bacterial artificial chromosome (BAC) clones, we show that it is composed of over 81% transposable elements, 77% of which are long terminal repeat (LTR) retrotransposons. Moreover, the LTR retrotransposon fraction in BAC clones harboring genes is disproportionately composed of chromodomain-containing Gypsy LTR retrotransposons ('chromoviruses'), and the majority of the intact chromoviruses contain tandem chromodomain duplications. We show that there is a bias in the efficacy of homologous recombination in removing LTR retrotransposon DNA, thereby providing insight into the mechanisms associated with transposable element (TE) composition in the sunflower genome. We also show that the vast majority of observed LTR retrotransposon insertions have likely occurred since the origin of this species, providing further evidence that biased LTR retrotransposon activity has played a major role in shaping the chromatin and DNA landscape of the sunflower genome. Although our findings on LTR retrotransposon age and structure could be influenced by the selection of the BAC clones analyzed, a global analysis of random sequence reads indicates that the evolutionary patterns described herein apply to the sunflower genome as a whole.

QTL mapping reveals the genetic architecture of loci affecting pre- and post-zygotic isolating barriers in Louisiana Iris.

BACKGROUND: Hybridization among Louisiana Irises has been well established and the genetic architecture of reproductive isolation is known to affect the potential for and the directionality of introgression between taxa. Here we use co-dominant markers to identify regions where QTL are located both within and between backcross maps to compare the genetic architecture of reproductive isolation and fitness traits across treatments and years. RESULTS: QTL mapping was used to elucidate the genetic architecture of reproductive isolation between Iris fulva and Iris brevicaulis. Homologous co-dominant EST-SSR markers scored in two backcross populations between I. fulva and I. brevicaulis were used to generate genetic linkage maps. These were used as the framework for mapping QTL associated with variation in 11 phenotypic traits likely responsible for reproductive isolation and fitness. QTL were dispersed throughout the genome, with the exception of one region of a single linkage group (LG) where QTL for flowering time, sterility, and fruit production clustered. In most cases, homologous QTL were not identified in both backcross populations, however, homologous QTL for flowering time, number of growth points per rhizome, number of nodes per inflorescence, and number of flowers per node were identified on several linkage groups. CONCLUSIONS: Two different traits affecting reproductive isolation, flowering time and sterility, exhibit different genetic architectures, with numerous QTL across the Iris genome controlling flowering time and fewer, less distributed QTL affecting sterility. QTL for traits affecting fitness are largely distributed across the genome with occasional overlap, especially on LG 4, where several QTL increasing fitness and decreasing sterility cluster. Given the distribution and effect direction of QTL affecting reproductive isolation and fitness, we have predicted genomic regions where introgression may be more likely to occur (those regions associated with an increase in fitness and unlinked to loci controlling reproductive isolation) and those that are less likely to exhibit introgression (those regions linked to traits decreasing fitness and reproductive isolation).

Genetic analysis of floral symmetry in Van Gogh's sunflowers reveals independent recruitment of CYCLOIDEA genes in the Asteraceae.

The genetic basis of floral symmetry is a topic of great interest because of its effect on pollinator behavior and, consequently, plant diversification. The Asteraceae, which is the largest family of flowering plants, is an ideal system in which to study this trait, as many species within the family exhibit a compound inflorescence containing both bilaterally symmetric (i.e., zygomorphic) and radially symmetric (i.e., actinomorphic) florets. In sunflower and related species, the inflorescence is composed of a single whorl of ray florets surrounding multiple whorls of disc florets. We show that in double-flowered (dbl) sunflower mutants (in which disc florets develop bilateral symmetry), such as those captured by Vincent van Gogh in his famous nineteenth-century sunflower paintings, an insertion into the promoter region of a CYCLOIDEA (CYC)-like gene (HaCYC2c) that is normally expressed specifically in WT rays is instead expressed throughout the inflorescence, presumably resulting in the observed loss of actinomorphy. This same gene is mutated in two independent tubular-rayed (tub) mutants, though these mutations involve apparently recent transposon insertions, resulting in little or no expression and radialization of the normally zygomorphic ray florets. Interestingly, a phylogenetic analysis of CYC-like genes from across the family suggests that different paralogs of this fascinating gene family have been independently recruited to specify zygomorphy in different species within the Asteraceae.

SNP discovery and development of a high-density genotyping array for sunflower.

Recent advances in next-generation DNA sequencing technologies have made possible the development of high-throughput SNP genotyping platforms that allow for the simultaneous interrogation of thousands of single-nucleotide polymorphisms (SNPs). Such resources have the potential to facilitate the rapid development of high-density genetic maps, and to enable genome-wide association studies as well as molecular breeding approaches in a variety of taxa. Herein, we describe the development of a SNP genotyping resource for use in sunflower (Helianthus annuus L.). This work involved the development of a reference transcriptome assembly for sunflower, the discovery of thousands of high quality SNPs based on the generation and analysis of ca. 6 Gb of transcriptome re-sequencing data derived from multiple genotypes, the selection of 10,640 SNPs for inclusion in the genotyping array, and the use of the resulting array to screen a diverse panel of sunflower accessions as well as related wild species. The results of this work revealed a high frequency of polymorphic SNPs and relatively high level of cross-species transferability. Indeed, greater than 95% of successful SNP assays revealed polymorphism, and more than 90% of these assays could be successfully transferred to related wild species. Analysis of the polymorphism data revealed patterns of genetic differentiation that were largely congruent with the evolutionary history of sunflower, though the large number of markers allowed for finer resolution than has previously been possible.

Downy mildew (Pl ( 8 ) and Pl ( 14 )) and rust (R ( Adv )) resistance genes reside in close proximity to tandemly duplicated clusters of non-TIR-like NBS-LRR-encoding genes on sunflower chromosomes 1 and 13.

Nucleotide binding site-leucine rich repeat (NBS-LRR) proteins are encoded by a ubiquitous gene family in sunflower and frequently harbor disease resistance genes. We investigated NBS-LRR-encoding resistance gene candidates (RGCs) flanking the downy mildew resistance genes Pl ( 8 ) and Pl ( 14 ) and the rust resistance gene R ( Adv ), which map on the NBS-LRR clusters of linkage groups 1 and 13 in sunflower genome. We shotgun sequenced bacterial artificial chromosome (BAC) clones proximal to Pl ( 8 ), Pl ( 14 ) , and R ( Adv ) and identified seven novel non-Toll/interleukin-1 receptor (TIR)-like NBS-LRR RGCs, which clustered with previously identified RGCs of linkage group 13 but were phylogenetically distant from the TIR- and non-TIR-NBS-LRR-encoding superfamilies of sunflower. Six of the seven predicted RGCs have intact open reading frames and reside in genomic segments with abundant transposable elements. The genomic localization and sequence similarity of the novel non-TIR-like predicted RGCs suggests that they originated from tandem duplications. RGCs in the proximity of Pl ( 8 ) and R ( Adv ) were likely introgressed from silverleaf sunflower genome, where the RGC cluster of linkage group 13 is duplicated in two independent chromosomes that have different architecture and level of recombination from the respective common sunflower chromosomes.

Transmission ratio distortion results in asymmetric introgression in Louisiana Iris.

BACKGROUND: Linkage maps are useful tools for examining both the genetic architecture of quantitative traits and the evolution of reproductive incompatibilities. We describe the generation of two genetic maps using reciprocal interspecific backcross 1 (BC1) mapping populations from crosses between Iris brevicaulis and Iris fulva. These maps were constructed using expressed sequence tag (EST)- derived codominant microsatellite markers. Such a codominant marker system allowed for the ability to link the two reciprocal maps, and compare patterns of transmission ratio distortion observed between the two. RESULTS: Linkage mapping resulted in markers that coalesced into 21 linkage groups for each of the reciprocal backcross maps, presumably corresponding to the 21 haploid chromosomes of I. brevicaulis and I. fulva. The composite map was 1190.0-cM long, spanned 81% of the I. brevicaulis and I. fulva genomes, and had a mean density of 4.5 cM per locus. Transmission ratio distortion (TRD) was observed in 138 (48.5%) loci distributed in 19 of the 21 LGs in BCIB, BCIF, or both BC1 mapping populations. Of the distorted markers identified, I. fulva alleles were detected at consistently higher-than-expected frequencies in both mapping populations. CONCLUSIONS: The observation that I. fulva alleles are overrepresented in both mapping populations suggests that I. fulva alleles are favored to introgress into I. brevicaulis genetic backgrounds, while I. brevicaulis alleles would tend to be prevented from introgressing into I. fulva. These data are consistent with the previously observed patterns of introgression in natural hybrid zones, where I. fulva alleles have been consistently shown to introgress across species boundaries.

Pleiotropy of the branching locus (B) masks linked and unlinked quantitative trait loci affecting seed traits in sunflower.

The discovery of unbranched, monocephalic natural variants was pivotal for the domestication of sunflower (Helianthus annuus L.). The branching locus (B), one of several loci apparently targeted by aboriginal selection for monocephaly, pleiotropically affects plant, seed and capitula morphology and, when segregating, confounds the discovery of favorable alleles for seed yield and other traits. The present study was undertaken to gain deeper insights into the genetics of branching and seed traits affected by branching. We produced an unbranched hybrid testcross recombinant inbred line (TC-RIL) population by crossing branched (bb) and unbranched (BB) RILs to an unbranched (BB) tester. The elimination of branching concomitantly eliminated a cluster of B-linked seed trait quantitative trait loci (QTL) identified by RIL per se testing. We identified a seed oil content QTL linked in repulsion and a 100-seed weight QTL linked in coupling to the B locus and additional unlinked QTL, previously masked by B-locus pleiotropy. Genomic segments flanking the B locus harbor multiple loci for domestication and post-domestication traits, the effects of which are masked by B-locus pleiotropy in populations segregating for branching and can only be disentangled by genetic analyses in unbranched populations. QTL analyses of NILs carrying wild B alleles substantiated the pleiotropic effects of the B locus. The effect of the B locus on branching was masked by the effects of wild alleles at independent branching loci in hybrids between monocephalic domesticated lines and polycephalic wild ecotypes; hence, the B locus appears to be necessary, but not sufficient, for monocephaly in domesticated sunflower.

Asymmetric introgressive hybridization among louisiana iris species.

In this review, we discuss findings from studies carried out over the past 20+ years that document the occurrence of asymmetric introgressive hybridization in a plant clade. In particular, analyses of natural and experimental hybridization have demonstrated the consistent introgression of genes from Iris fulva into both Iris brevicaulis and Iris hexagona. Furthermore, our analyses have detected certain prezygotic and postzygotic barriers to reproduction that appear to contribute to the asymmetric introgression. Finally, our studies have determined that a portion of the genes transferred apparently affects adaptive traits.

Development, polymorphism, and cross-taxon utility of EST-SSR markers from safflower (Carthamus tinctorius L.).

Due to their highly polymorphic and codominant nature, simple-sequence repeat (SSR) markers are a common choice for assaying genetic diversity and genetic mapping. In this paper, we describe the generation of an expressed-sequence tag (EST) collection for the oilseed crop safflower and the subsequent development of EST-SSR markers for the genetic analysis of safflower and related species. We assembled 40,874 reads into 19,395 unigenes, of which 4,416 (22.8%) contained at least one SSR. Primer pairs were developed and tested for 384 of these loci, resulting in a collection of 104 polymorphic markers that amplify reliably across 27 accessions (3 species) of the genus Carthamus. These markers exhibited a high level of polymorphism, with an average of 6.0 +/- 0.4 alleles per locus and an average gene diversity of 0.54 +/- 0.03 across Carthamus species. In terms of cross-taxon transferability, 50% of these primer pairs produced an amplicon in at least one other species in the Asteraceae, and 28% produced an amplicon in at least one species outside the safflower subfamily (i.e., lettuce, sunflower, and/or Gerbera). These markers represent a valuable resource for the genetic analysis of safflower and related species, and also have the potential to facilitate comparative map-based analyses across a broader array of taxa within the Asteraceae.

EST and EST-SSR marker resources for Iris.

BACKGROUND: Limited DNA sequence and DNA marker resources have been developed for Iris (Iridaceae), a monocot genus of 200-300 species in the Asparagales, several of which are horticulturally important. We mined an I. brevicaulis-I. fulva EST database for simple sequence repeats (SSRs) and developed ortholog-specific EST-SSR markers for genetic mapping and other genotyping applications in Iris. Here, we describe the abundance and other characteristics of SSRs identified in the transcript assembly (EST database) and the cross-species utility and polymorphisms of I. brevicaulis-I. fulva EST-SSR markers among wild collected ecotypes and horticulturally important cultivars. RESULTS: Collectively, 6,530 ESTs were produced from normalized leaf and root cDNA libraries of I. brevicaulis (IB72) and I. fulva (IF174), and assembled into 4,917 unigenes (1,066 contigs and 3,851 singletons). We identified 1,447 SSRs in 1,162 unigenes and developed 526 EST-SSR markers, each tracing a different unigene. Three-fourths of the EST-SSR markers (399/526) amplified alleles from IB72 and IF174 and 84% (335/399) were polymorphic between IB25 and IF174, the parents of I. brevicaulis x I. fulva mapping populations. Forty EST-SSR markers were screened for polymorphisms among 39 ecotypes or cultivars of seven species - 100% amplified alleles from wild collected ecotypes of Louisiana Iris (I.brevicaulis, I.fulva, I. nelsonii, and I. hexagona), whereas 42-52% amplified alleles from cultivars of three horticulturally important species (I. pseudacorus, I. germanica, and I. sibirica). Ecotypes and cultivars were genetically diverse - the number of alleles/locus ranged from two to 18 and mean heterozygosity was 0.76. CONCLUSION: Nearly 400 ortholog-specific EST-SSR markers were developed for comparative genetic mapping and other genotyping applications in Iris, were highly polymorphic among ecotypes and cultivars, and have broad utility for genotyping applications within the genus.

A genomic scan for selection reveals candidates for genes involved in the evolution of cultivated sunflower (Helianthus annuus).

Genomic scans for selection are a useful tool for identifying genes underlying phenotypic transitions. In this article, we describe the results of a genome scan designed to identify candidates for genes targeted by selection during the evolution of cultivated sunflower. This work involved screening 492 loci derived from ESTs on a large panel of wild, primitive (i.e., landrace), and improved sunflower (Helianthus annuus) lines. This sampling strategy allowed us to identify candidates for selectively important genes and investigate the likely timing of selection. Thirty-six genes showed evidence of selection during either domestication or improvement based on multiple criteria, and a sequence-based test of selection on a subset of these loci confirmed this result. In view of what is known about the structure of linkage disequilibrium across the sunflower genome, these genes are themselves likely to have been targeted by selection, rather than being merely linked to the actual targets. While the selection candidates showed a broad range of putative functions, they were enriched for genes involved in amino acid synthesis and protein catabolism. Given that a similar pattern has been detected in maize (Zea mays), this finding suggests that selection on amino acid composition may be a general feature of the evolution of crop plants. In terms of genomic locations, the selection candidates were significantly clustered near quantitative trait loci (QTL) that contribute to phenotypic differences between wild and cultivated sunflower, and specific instances of QTL colocalization provide some clues as to the roles that these genes may have played during sunflower evolution.

SSRs and INDELs mined from the sunflower EST database: abundance, polymorphisms, and cross-taxa utility.

Simple sequence repeats (SSRs) are abundant and frequently highly polymorphic in transcribed sequences and widely targeted for marker development in eukaryotes. Sunflower (Helianthus annuus) transcript assemblies were built and mined to identify SSRs and insertions-deletions (INDELs) for marker development, comparative mapping, and other genomics applications in sunflower. We describe the spectrum and frequency of SSRs identified in the sunflower EST database, a catalog of 16,643 EST-SSRs, a collection of 484 EST-SSR and 43 EST-INDEL markers developed from common sunflower ESTs, polymorphisms of the markers among the parents of several intraspecific and interspecific mapping populations, and the transferability of the markers to closely and distantly related species in the Compositae. Of 17,904 unigenes in the transcript assembly, 1,956 (10.9%) harbored one or more SSRs with repeat counts of n > or = 5. EST-SSR markers were 1.6-fold more polymorphic among exotic than elite genotypes and 0.7-fold less polymorphic than non-genic SSR markers. Of 466 EST-SSR or INDEL markers screened for cross-species amplification and polymorphisms, 413 (88.6%) amplified alleles from one or more wild species (H. argophyllus, H. tuberosus, H. anomalus, H. paradoxus, and H. deserticola), whereas 69 (14.8%) amplified alleles from safflower (Carthamus tinctorius) and 67 (14.4%) amplified alleles from lettuce (Lactuca sativa); hence, only a fraction were transferable to distantly related genera in the Compositae, whereas most were transferable to wild relatives of H. annuus. Several thousand additional SSRs were identified in the EST database and supply a wealth of templates for EST-SSR marker development in sunflower.

Single nucleotide polymorphisms and linkage disequilibrium in sunflower.

Genetic diversity in modern sunflower (Helianthus annuus L.) cultivars (elite oilseed inbred lines) has been shaped by domestication and breeding bottlenecks and wild and exotic allele introgression(-)the former narrowing and the latter broadening genetic diversity. To assess single nucleotide polymorphism (SNP) frequencies, nucleotide diversity, and linkage disequilibrium (LD) in modern cultivars, alleles were resequenced from 81 genic loci distributed throughout the sunflower genome. DNA polymorphisms were abundant; 1078 SNPs (1/45.7 bp) and 178 insertions-deletions (INDELs) (1/277.0 bp) were identified in 49.4 kbp of DNA/genotype. SNPs were twofold more frequent in noncoding (1/32.1 bp) than coding (1/62.8 bp) sequences. Nucleotide diversity was only slightly lower in inbred lines ( = 0.0094) than wild populations ( = 0.0128). Mean haplotype diversity was 0.74. When extraploted across the genome ( approximately 3500 Mbp), sunflower was predicted to harbor at least 76.4 million common SNPs among modern cultivar alleles. LD decayed more slowly in inbred lines than wild populations (mean LD declined to 0.32 by 5.5 kbp in the former, the maximum physical distance surveyed), a difference attributed to domestication and breeding bottlenecks. SNP frequencies and LD decay are sufficient in modern sunflower cultivars for very high-density genetic mapping and high-resolution association mapping.

Ty3/gypsy-like retrotransposon knockout of a 2-methyl-6-phytyl-1,4-benzoquinone methyltransferase is non-lethal, uncovers a cryptic paralogous mutation, and produces novel tocopherol (vitamin E) profiles in sunflower.

The m (Tph(1)) mutation partially disrupts the synthesis of alpha-tocopherol (vitamin E) in sunflower (Helianthus annuus L.) seeds and was predicted to disrupt a methyltransferase activity necessary for the synthesis of alpha- and gamma-tocopherol. We identified and isolated two 2-methyl-6-phytyl-1,4-benzoquinone/2-methyl-6-solanyl-1,4-benzoquinone methyltransferase (MPBQ/MSBQ-MT) paralogs from sunflower (MT-1 and MT-2), resequenced MT-1 and MT-2 alleles from wildtype (m(+) m(+)) and mutant (m m) inbred lines, identified m as a non-lethal knockout mutation of MT-1 caused by the insertion of a 5.2 kb Ty3/gypsy-like retrotransposon in exon 1, and uncovered a cryptic codominant mutation (d) in a wildtype x mutant F(2) population predicted to be segregating for the m mutation only. MT-1 and m cosegregated and mapped to linkage group 1 and MT-1 was not transcribed in mutant homozygotes (m m). The m locus was epistatic to the d locus--the d locus had no effect in m(+) m(+) and m(+) m individuals, but significantly increased beta-tocopherol percentages in m m individuals. MT-2 and d cosegregated, MT-2 alleles isolated from mutant homozygotes (d d) carried a 30 bp insertion at the start of the 5'-UTR, and MT-2 was more strongly transcribed in seeds and leaves of wildtype (d(+) d(+)) than mutant (d d) homozygotes (transcripts were 2.2- to 5.0-fold more abundant in the former than the latter). The double mutant (m m d d) was non-lethal and produced 24-45% alpha- and 55-74% beta-tocopherol (the wildtype produced 96% alpha- and 4% beta-tocopherol). MT-2 compensated for the loss of the MT-1 function, and the MT-2 mutation profoundly affected the synthesis of tocopherols without adversely affecting the synthesis of plastoquinone crucial for normal plant growth and development.

Three non-allelic epistatically interacting methyltransferase mutations produce novel tocopherol (vitamin E) profiles in sunflower.

Wildtype sunflower (Helianthus annuus L.) seeds are a rich source of alpha-tocopherol (vitamin E). The g = Tph(2) mutation disrupts the synthesis of alpha-tocopherol, enhances the synthesis of gamma-tocopherol, and was predicted to knock out a gamma-tocopherol methyltransferase (gamma-TMT) necessary for the synthesis of alpha-tocopherol in sunflower seeds--wildtype (g(+) g(+)) lines accumulated > 90% alpha-tocopherol, whereas mutant (g g) lines accumulated > 90% gamma-tocopherol. We identified and isolated two gamma-TMT paralogs (gamma-TMT-1 and gamma-TMT-2). Both mapped to linkage group 8, cosegregated with the g locus, and were transcribed in developing seeds of wildtype lines. The g mutation greatly decreased gamma-TMT-1 transcription, caused alternative splicing of gamma-TMT-1, disrupted gamma-TMT-2 transcription, and knocked out one of two transcription initiation sites identified in the wildtype; gamma-TMT transcription was 36 to 51-fold greater in developing seeds of wildtype (g(+) g(+)) than mutant (g g) lines. F(2) populations (B109 x LG24 and R112 x LG24) developed for mapping the g locus segregated for a previously unidentified locus (d). B109, R112, and LG24 were homozygous for a null mutation (m = Tph(1)) in MT-1, one of two 2-methyl-6-phytyl-1,4-benzoquinone/2-methyl-6-solanyl-1,4-benzoquinone methyltransferase (MPBQ/MSBQ-MT) paralogs identified in sunflower. The d mutations segregating in B109 x LG24 and R112 x LG24 were allelic to a cryptic mutation identified in the other MPBQ/MSBQ-MT paralog (MT-2) and disrupted the synthesis of alpha- and gamma-tocopherol in F(2) progeny carrying m or g mutations--m m g(+) g(+) d d homozygotes accumulated 41.5% alpha- and 58.5% beta-T, whereas m m g g d d homozygotes accumulated 58.1% gamma- and 41.9% delta-T. MT-2 cosegregated with d and mapped to linkage group 4. Hence, novel tocopherol profiles are produced in sunflower seed oil by three non-allelic epistatically interacting methyltransferase mutations.

Extensive chromosomal repatterning and the evolution of sterility barriers in hybrid sunflower species.

New species may arise via hybridization and without a change in ploidy. This process, termed homoploid hybrid speciation, is theoretically difficult because it requires the development of reproductive barriers in sympatry or parapatry. Theory suggests that isolation may arise through rapid karyotypic evolution and/or ecological divergence of hybrid neospecies. Here, we investigate the role of karyotypic change in homoploid hybrid speciation by generating detailed genetic linkage maps for three hybrid sunflower species, Helianthus anomalus, H. deserticola, and H. paradoxus, and comparing these maps to those previously generated for the parental species, H. annuus and H. petiolaris. We also conduct a quantitative trait locus (QTL) analysis of pollen fertility in a BC2 population between the parental species and assess levels of pollen and seed fertility in all cross-combinations of the hybrid and parental species. The three hybrid species are massively divergent from their parental species in karyotype; gene order differences were observed for between 9 and 11 linkage groups (of 17 total), depending on the comparison. About one-third of the karyoypic differences arose through the sorting of chromosomal rearrangements that differentiate the parental species, but the remainder appear to have arisen de novo (six breakages/six fusions in H. anomalus, four breakages/three fusions in H. deserticola, and five breakages/five fusions in H. paradoxus). QTL analyses indicate that the karyotypic differences contribute to reproductive isolation. Nine of 11 pollen viability QTL occur on rearranged chromosomes and all but one map close to a rearrangement breakpoint. Finally, pollen and seed fertility estimates for F1's between the hybrid and parental species fall below 11%, which is sufficient for evolutionary independence of the hybrid neospecies.

Comparative mapping and rapid karyotypic evolution in the genus helianthus.

Comparative genetic linkage maps provide a powerful tool for the study of karyotypic evolution. We constructed a joint SSR/RAPD genetic linkage map of the Helianthus petiolaris genome and used it, along with an integrated SSR genetic linkage map derived from four independent H. annuus mapping populations, to examine the evolution of genome structure between these two annual sunflower species. The results of this work indicate the presence of 27 colinear segments resulting from a minimum of eight translocations and three inversions. These 11 rearrangements are more than previously suspected on the basis of either cytological or genetic map-based analyses. Taken together, these rearrangements required a minimum of 20 chromosomal breakages/fusions. On the basis of estimates of the time since divergence of these two species (750,000-1,000,000 years), this translates into an estimated rate of 5.5-7.3 chromosomal rearrangements per million years of evolution, the highest rate reported for any taxonomic group to date.