Shoot development in many higher plant species is characterized by phase change, where meristems and organs transition from one set of identities to another. The transition from a juvenile to adult leaf identity in maize is regulated by the APETALA2-like gene glossy15 (gl15). We demonstrate here that increasing gl15 activity in transgenic maize not only increases the number of leaves expressing juvenile traits, but also delays the onset of reproductive development, indicating that gl15 plays a primary role in the maintenance of the juvenile phase. We also show that the accumulation of a maize microRNA homologous to miR172 increases during shoot development and mediates gl15 mRNA degradation. These data indicate that vegetative phase change in maize is regulated by the opposing actions of gl15 and miR172, with gl15 maintaining the juvenile phase and miR172 promoting the transition to the adult phase by down-regulation of gl15. Our results also suggest that the balance of activities between APETALA2-like genes and miR172 may be a general mechanism for regulating vegetative phase change in higher plants.juvenile-to-adult transition ͉ flowering time V egetative phase change has been characterized in many plant species (1, 2). The most obvious features that distinguish the basal juvenile and upper adult phases are the capacity for reproductive development (an adult trait) and heteroblasty, where leaves with distinct physiological and morphological identities are produced during shoot development (3, 4). Studies of vegetative phase change suggest that this process is regulated independently from the transition to reproductive development, although there may be overlap and coordination among vegetative and reproductive programs (5).In maize (Zea mays L.), the basal juvenile leaves (the first five or six in most genotypes) differ from upper adult leaves in their expression of a suite of epidermal cell characteristics, including epicuticular waxes, cell wall features, and the presence of specialized cell types such as leaf hairs. Mutations in at least seven maize genes and the gibberellic acid class of plant growth regulators have been shown to alter the relative expression of juvenile and adult leaf identity (2). Among these genes is glossy15 (gl15), an APETALA2-like gene that is expressed in juvenile leaves and plays a central role in regulating the epidermal cell traits that define leaf identity (6-8).The progressive nature of vegetative phase change in maize and other plant species suggests that this process is regulated by the relative levels of both juvenile and adult regulatory factors (6, 7). Other than gl15 being required to promote juvenile leaf identity in maize, little is known about the factors controlling vegetative phase change or their regulation. Recently, two studies in Arabidopsis have demonstrated that the microRNA miR172 antagonizes the activity of AP2 and a specific subset of AP2-like genes to regulate floral organ identity and flowering time (9, 10). The target sequence for miR172 is also present i...

Soybean [Glycine max (L.) Merr.] is primarily grown as a source of protein and oil. A quantitative trait locus (QTL) controlling seed protein concentration was previously mapped to linkage group (LG) I of soybean. The objectives of this study were to fine map the QTL and to determine if additional recombination could reduce the inverse phenotypic relationship between seed protein concentration and yield and oil concentration. The fine mapping was done with two sets of backcross populations that were tested in the field and with genetic markers. These populations were developed by the introgression of a high protein allele from the Glycine soja Sieb and Zucc. plant introduction (PI) 468916 into the genetic background of the breeding line A81–356022. The first set (Set 1) included three populations of backcross‐four (BC4) lines, and the second set (Set 2) included four populations of BC5 lines. The populations segregated for different segments of the genomic region where the QTL maps. Tests of the two sets of populations resulted in the localization of the QTL for protein and oil to a 3‐cM interval between the simple sequence repeat (SSR) marker Satt239 and the amplified fragment length polymorphism (AFLP) marker ACG9b. The results from the agronomic trait evaluations were inconsistent, making it difficult to definitively conclude whether the protein QTL controls these other traits through pleiotropy.

staining. However, these visualization methods require either expensive or hazardous radioactive chemicals and Microsatellite DNA markers are widely used in genetic research.are time-consuming. Electrophoresis with MetaPhor Their use, however, can be costly and throughput is sometimes limited. The objective of this paper is to introduce a simple, low-cost, high-agarose gels (Cambrex Corporation, East Rutherford, throughput system that detects amplification products from microsa-NJ) has been used to separate alleles of microsatellite tellite markers by nondenaturing polyacrylamide gel electrophoresis. markers, but the resolution is lower than nondenaturingThis system is capable of separating DNA fragments that differ by polyacrylamide gels and the cost is currently five times as little as two base pairs. The electrophoresis unit holds two vertical more than that of nondenaturing polyacrylamide gels. 100-sample gels allowing standards and samples from a 96-well plateCapillary electrophoresis also has been used to deterto be analyzed on a single gel. DNA samples are stained during mine length polymorphisms of microsatellite markers electrophoresis by ethidium bromide in the running buffer. In addi- (Marino et al., 1995), but this method requires sophistition, one of the gel plates is UV-transparent so that gels can be cated instruments and fluorescently tagged primers, photographed immediately after electrophoresis without disassemwhich are expensive. Here we describe an inexpensive bling the gel-plate sandwich. Electrophoresis runs are generally less than two hours. The cost per gel, excluding PCR cost, is currently and relatively high-throughput system developed for the estimated at about $2.60, or less than $0.03 per data point. This system purpose of genotyping with microsatellite markers. has been used successfully with soybean [Glycine max (L.) Merr.] and wheat (Triticum aestivum L.) microsatellite markers and could 1828

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The production of resistant soybean [Glycine max (L.) Merr.] cultivars is the most effective means for controlling losses from soybean cyst nematode (SCN) (Heterodera glycines Ichinohe). The major resistance gene in most SCN resistance sources is rhg1, which has been mapped as a quantitative trait locus onto linkage group G. Our objective was to determine whether the SCN resistance sources PI 437654 and PI 88788 have different functional alleles at rhg1 based on resistance phenotypes. Populations segregating for resistance alleles at rhg1 from both PI 88788 and PI 437654 and at Rhg4, a second SCN resistance gene from PI 437654, were developed. These populations were screened for resistance to the H. glycines inbred isolates PA3 (HG type 7) and TN14 (HG type 1.2.5.7) in the greenhouse and evaluated with molecular markers linked to both rhg1 and Rhg4. Each isolate test was repeated, and the evaluations were done on a single-plant and a line-mean basis in Test 1, and solely on a single-plant basis in Test 2. Across two tests with the TN14 isolate, plants with the PI 437654 allele for a marker linked to rhg1 had significantly (P<0.0001) less SCN reproduction than plants carrying the PI 88788 allele. A marker linked to Rhg4, however, was not significantly associated with resistance to TN14. Across two tests with the PA3 isolate, alleles of rhg1 from both sources gave a resistant reaction, although plants homozygous for the PI 88788 allele had significantly (P<0.05) greater resistance than plants with the PI 437654 allele. The marker allele from PI 437654 linked to Rhg4 was significantly (P<0.0005) associated with greater resistance than the PI 88788 allele in both PA3 tests, and resistance was dominant. There was a significant interaction between alleles at rhg1 and Rhg4 in both PA3 tests. These results suggest that PI 437654 and PI 88788 each have a different functional SCN resistance allele at or close to rhg1. These allelic differences have implications that breeders should consider before incorporation into cultivars.

Autonomous chromosomes are generated in yeast (yeast artificial chromosomes) and human fibrosarcoma cells (human artificial chromosomes) by introducing purified DNA fragments that nucleate a kinetochore, replicate, and segregate to daughter cells. These autonomous minichromosomes are convenient for manipulating and delivering DNA segments containing multiple genes. In contrast, commercial production of transgenic crops relies on methods that integrate one or a few genes into host chromosomes; extensive screening to identify insertions with the desired expression level, copy number, structure, and genomic location; and long breeding programs to produce varieties that carry multiple transgenes. As a step toward improving transgenic crop production, we report the development of autonomous maize minichromosomes (MMCs). We constructed circular MMCs by combining DsRed and nptII marker genes with 7–190 kb of genomic maize DNA fragments containing satellites, retroelements, and/or other repeats commonly found in centromeres and using particle bombardment to deliver these constructs into embryogenic maize tissue. We selected transformed cells, regenerated plants, and propagated their progeny for multiple generations in the absence of selection. Fluorescent in situ hybridization and segregation analysis demonstrated that autonomous MMCs can be mitotically and meiotically maintained. The MMC described here showed meiotic segregation ratios approaching Mendelian inheritance: 93% transmission as a disome (100% expected), 39% transmission as a monosome crossed to wild type (50% expected), and 59% transmission in self crosses (75% expected). The fluorescent DsRed reporter gene on the MMC was expressed through four generations, and Southern blot analysis indicated the encoded genes were intact. This novel approach for plant transformation can facilitate crop biotechnology by (i) combining several trait genes on a single DNA fragment, (ii) arranging genes in a defined sequence context for more consistent gene expression, and (iii) providing an independent linkage group that can be rapidly introgressed into various germplasms.

that Race 3 SCN resistance in PI 88788 is inherited by three genes, with one recessive and two dominant. The Soybean cyst nematode (SCN) (Heterodera glycines Ichinohe) is genetic evidence indicates that one of the dominant genes one of the most destructive soybean [Glycine max (L.) Merr.] pests is at a previously unreported locus which was designated worldwide. The most common source of SCN resistance used in soybean breeding in the northern USA is PI 88788. Previous research Rhg5 (Rao Arelli, 1994) the second gene is Rhg4, which has shown that PI 88788 carries a major quantitative trait locus (QTL) maps close to the i gene (Matson and Williams, 1965), conferring SCN resistance on linkage group (LG) G, which is believed and the recessive gene is rhg2. Genetic mapping efforts to be rhg1. The objective of our research was to map and confirm have since shown that PI 88788 has a major QTL on additional SCN resistance QTL in Bell, a cultivar with resistance LG G (Concibido et al., 1997), and a second minor QTL from PI 88788. One hundred four F 4 -derived lines (F 4 population) on LG C2 (Diers et al., 1997a). The QTL on LG G maps developed from crossing the cultivars Bell and Colfax were tested for to the same region where a major resistance locus was associations between 54 molecular markers and resistance to SCN mapped in PI 437654 (Webb et al., 1995), Peking, PI populations PA3 (HG type 7, race 3) and PA14 (HG type 1.3.5.6.7, 90763, PI 89772, and PI 209332 (Concibido et al., 1997; race 14). Three populations of near isogenic lines (NILs) were devel-Concibido et al., 1996; Yue et al., 2001). The resistance oped from F 4 plants heterozygous for a region on LG J where a significant QTL was identified in the F 4 population. The NIL popula-gene in this region has been designated rhg1 in the tions were tested with genetic markers and also for resistance to both literature and Cregan et al. (1999b) reported a linkage SCN populations. In the F 4 population, SCN resistance QTL were of 0.4 centimorgans (cM) between the simple sequence identified at both rhg1 and on LG J. The LG J QTL was confirmed repeat (SSR) marker Satt309 and rhg1 in crosses with in NIL populations and was given the confirmed QTL designation Peking and PI 209332 as sources of SCN resistance. cqSCN-003. The effect of cqSCN-003 was diminished in the NIL Although many QTL have been mapped in soybean, populations compared to the F 4 population. This was at least partially few have been confirmed in additional populations in the result of segregation distortion in the F 4 population between the the same or different genetic backgrounds. The confirregion containing rhg1 and the region containing cqSCN-003. These mation of QTL after initial mapping is a critical step results show the importance of verifying QTL in confirmation populabefore the selection of the QTL with markers in breedtions to estimate accurately their effects.ing programs. Near isogenic line populations are particularly useful for QTL confirmation because they are developed to segregate for QTL ...

ping of all three resistance genes to a region near the simple sequence repeat (SSR) markers Satt431 and Satt244 Many soybean [Glycine max (L.) Merr.] genotypes that carry resison LG J (Bachman et al., 2001; Bachman and Nickell, tance to soybean cyst nematode (Heterodera glycines Ichinohe) (SCN)