Seed trait evaluation of <Emphasis Type="Italic">Gossypium barbadense</Emphasis> L. chromosomes/arms in a <Emphasis Type="Italic">G. hirsutum</Emphasis> L. background

Investigation of cotton nutritional components is important because its seeds provide a useful nutritional profile and can possibly serve as a biofuel resource. In this study, five cultivars, 13 cotton chromosome substitution (CS-B) lines, their donor parent, '3-79', and their recurrent parent, 'TM-1', were evaluated for seed traits over four environments. A mixed linear model approach with the jackknife method was employed to estimate variance components and to predict genotypic effects for each seed trait. Genotypic effects were more important than genotype by environment interaction for all seed traits. Chromosome associations with these seed traits were detected using the comparative method by comparing the differences between each CS-B line and TM-1. For example, chromosome 4 of 3-79 in TM-1 background was associated with reduced seed index (SI), embryo percentage, protein percentage while associated with increased seed oil percentage and seed fiber percentage. Other chromosome associations with these seed traits were also observed in this study. SI was highly correlated with three seed index traits: seed protein index, seed oil index (OI), and seed fiber index. Lint percentage, boll number, and lint yield were positively correlated with protein percentage while negatively correlated with SI and OI. SI and seed fiber content exhibited negative correlations with micronaire but positive correlations with fiber length and strength. Results suggested that agronomic traits and seed nutrition components can be improved simultaneously.     

Genetic control of agronomic traits in alfalfa (<Emphasis Type="Italic">M. sativa</Emphasis> ssp. <Emphasis Type="Italic">sativa</Emphasis> L.)

The objective of this study was to develop diallel population hybrids by crossing selected germplasm and to determine the gene effects and genetic control of yield and yield components using diallel analysis. A complete diallel including reciprocals was made during 2003 and 2004 between five alfalfa cultivars of different geographic origin. For each pairwise cross, five plants were chosen at random from each of the two cultivars (~100 florets per plant) to obtain the F₁ generation. A spaced plant field was established in 2006 which included the five alfalfa cultivars (parents) and their 20 diallel hybrids (F₁). The results of the diallel analysis suggest that the genetic control of major agronomic traits is determined by both additive gene action (accumulation of frequency of desirable alleles represented by significant GCA effects) and nonadditive gene action (complementary gene interactions represented by significant SCA effects). This type of gene action expression in alfalfa also determines the way in which breeding is carried out and brings about changes in the methods used and has given rise to the idea of the semi-hybrid breeding of this crop. The concept involves: breeding alfalfas within the population, identification of heterotic germplasm, and the production of seed of the population hybrid (PH).     

Molecular mapping of <Emphasis Type="Italic">Pc68</Emphasis>, a crown rust resistance gene in <Emphasis Type="Italic">Avena</Emphasis><Emphasis Type="Italic">sativa</Emphasis>

Crown rust, which is caused by Puccinia coronata f. sp. avenae, P. Syd. & Syd., is the most destructive disease of cultivated oats (Avena sativa L.) throughout the world. Resistance to the disease that is based on a single gene is often short-lived because of the extremely great genetic diversity of P. coronata, which suggests that there is a need to develop oat cultivars with several resistance genes. This study aimed to identify amplified fragment length polymorphism AFLP markers that are linked to the major resistance gene, Pc68, and to amplify the F₆ genetic map from Pc68/5*Starter × UFRGS8. Seventy-eight markers with normal segregation were discovered and distributed in 12 linkage groups. The map covered 409.4 cM of the Avena sativa genome. Two AFLP markers were linked in repulsion to Pc68: U8PM22 and U8PM25, which flank the gene at 18.60 and 18.83 centiMorgans (cM), respectively. The marker U8PM25 is located in the linkage group 4_12 in the Kanota × Ogle reference oat population. These markers should be useful for transferring Pc68 to genotypes with good agronomic characteristics and for pyramiding crown rust resistance genes.     

<Emphasis Type="Italic">Rf4</Emphasis> has minor effects on the fertility restoration of wild abortive-type cytoplasmic male sterile <Emphasis Type="Italic">japonica</Emphasis> (<Emphasis Type="Italic">Oryza sativa</Emphasis>) lines

Wild abortive (WA)-type cytoplasmic male sterility (CMS) has been exclusively used for breeding three-line hybrid indica rice, but it has not been applied for generating japonica hybrids because of the difficulties related to breeding japonica restorer lines. Determining whether the major restorer-of-fertility (Rf) gene used for indica hybrids can efficiently restore the fertility of WA-type japonica CMS lines may be useful for breeding WA-type japonica restorer lines. In this study, japonica restorer lines for Chinsurah Boro II (BT)-type CMS exhibited varying abilities to restore the fertility of ‘WA-LiuqianxinA’, which is a WA-type japonica CMS line. Additionally, Rf genes for WA-type CMS were identified in the BT-type japonica restorers. Meanwhile, ‘C9083’, which is a BT-type japonica restorer, exhibited a limited ability to restore the fertility of WA-type japonica CMS lines, and a genetic analysis revealed that the fertility restoration was controlled by one locus. The Rf gene was mapped to an approximately 370-kb physical region and was identified as Rf4. Furthermore, Rf gene dosage effects and the temperature influenced the fertility restoration of WA-type japonica CMS lines. This study is the first to confirm that Rf4 has only minor effects on the fertility restoration of WA-type japonica CMS lines. These results may be relevant for the development of WA-type japonica hybrids.     

Genetic analysis of the fiber quality and yield traits in <Emphasis Type="Italic">G. hirsutum</Emphasis> background using chromosome segments substitution lines (CSSLs) from <Emphasis Type="Italic">Gossypium barbadense</Emphasis>

Developing chromosome segments substitution lines (CSSLs) is an effective method for broadening the cotton germplasm resource, and improving the fiber quality and yield traits. In this study, the 1054 F₂ individual plants and 116 F_2:3 lineages were generated from the two parents of MBI9749 and MBI9915 selected from BC₅F_3:5 lines which originated from hybridization of CCRI36 and Hai1, and advanced backcrossing and repeated selfing. Genotypes of the parents and F₂ population were analyzed. The results showed that 19 segments were introgressed for MBI9749 and 12 segments were introgressed for MBI9915, distributing on 17 linkage groups. The average background recovery rate to the recurrent parent CCRI36 was 96.70% for the two parents. An average of 16.46 segments was introgressed in F₂ population. The average recovery rate of 1054 individual plants was 96.85%, and the mean length of sea island introgression segments was 157.18 cM, accounting for 3.15% of detection length. QTL mapping analysis detected 22 QTLs associated with fiber quality and yield traits in the F₂ and F_2:3 populations. These QTLs distributed on seven chromosomes, and the phenotypic variation was explained ranging from 1.20 to 14.61%. Four stable QTLs were detected in F₂ and F_2:3 populations, simultaneously. We found that eight QTLs were in agreement with the previous research. Six QTL-clusters were identified for fiber quality and yield traits, in which five QTL-clusters were on chromosome20. The results indicated that most of QTL-clusters always improve the fiber quality and have negative additive effect for yield related traits. This study demonstrated that CSSLs provide basis for fine mapping of the fiber quality and yield traits in future, and could be efficiently used for pyramiding favourable alleles to develop the new germplasms for breeding by molecular marker-assisted selection.     

RDA derived <Emphasis Type="Italic">Oryza minuta</Emphasis>-specific clones to probe genomic conservation across <Emphasis Type="Italic">Oryza</Emphasis> and introgression into rice (<Emphasis Type="Italic">O. sativa</Emphasis> L.)

Molecular markers have been successfully used in rice breeding however available markers based on Oryza sativa sequences are not efficient to monitor alien introgression from distant genomes of Oryza. We developed O. minuta (2n = 48, BBCC)-specific clones comprising of 105 clones (266–715 bp) from the initial library composed of 1,920 clones against O. sativa by representational difference analysis (RDA), a subtractive cloning method and validated through Southern blot hybridization. Chromosomal location of O. minuta-specific clones was identified by hybridization with the genomic DNA of eight monosomic alien additional lines (MAALs). The 37 clones were located either on chromosomes 6, 7, or 12. Different hybridization patterns between O. minuta-specific clones and wild species such as O. punctata, O. officinalis, O. rhizomatis, O. australiensis, and O. ridleyi were observed indicating conservation of the O. minuta fragments across Oryza spp. A highly repetitive clone, OmSC45 hybridized with O. minuta and O. australiensis (EE), and was found in 6,500 and 9,000 copies, respectively, suggesting an independent and exponential amplification of the fragment in both species during the evolution of Oryza. Hybridization of 105 O. minuta specific clones with BB- and CC-genome wild Oryza species resulted in the identification of 4 BB-genome-specific and 14 CC-genome-specific clones. OmSC45 was identified as a fragment of RIRE1, an LTR-retrotransposon. Furthermore this clone was introgressed from O. minuta into the advanced breeding lines of O. sativa.     

Fine mapping of grain weight QTLs using near isogenic lines from a Cross between <Emphasis Type="Italic">Oryza sativa</Emphasis> and <Emphasis Type="Italic">O. grandiglumis</Emphasis>

In a previous study, we reported the grain weight QTL, tgw2 in the 150 F2:3 lines derived from a cross between Oryza sativa subssp. Japonica cv. Hwaseongbyeo and HG101. This QTL was confirmed in F4 lines (CR1242) segregating for the target region. For fine mapping of tgw2, one F₅ plant homozygous for the O. grandiglumis DNA in the target region was selected from CR1242 and crossed with Hwaseongbyeo to produce the F₂ and F₃ populations. QTL analysis using 490 F₂ plants confirmed the existence of tgw2 with an R ² value of 28.0%. This QTL explained 61.3% of the phenotypic variance for 1,000-grain weight in 64 F₃ lines. Substitution mapping with 47 F₃ lines and 74 F₄ plants with informative recombination breakpoints in the target region was carried out to narrow down the position of the tgw2. The result indicated that tgw2 was located in the 384-kb interval between two SSR markers, RM12813 and RM12836. Annotation data of BACs in this 384-kb region revealed that forty-five putative genes exist in this interval including the GW2 gene responsible for grain weight and width. Considering the position of the QTL tgw2, it appears that tgw2 is functionally related to the gene GW2. However, the possibility that another unknown mechanism might be responsible for regulation of grain weight at tgw2 cannot be ruled out. Four QTLs for grain length, grain width, and grain thickness were also located in the same interval suggesting that a single gene is involved in controlling these four traits. Substitution mapping also indicated that two QTLs for grain weight and culm length, tgw2 and cl2, were tightly linked.     

Identification and molecular cytology analysis of cold tolerance introgression lines derived from <Emphasis Type="Italic">Oryza sativa</Emphasis> L. mating with <Emphasis Type="Italic">O. rufipogon</Emphasis> Griff.

A previously constructed introgression lines (ILs) population including 239 lines was used to evaluate the cold-tolerant ability. The ILs with strong cold tolerance which were identified in the present study were used for further cytological and microsatellite (SSR) marker analyses. The results showed that the IL5243 and IL5335 had strong cold-tolerant ability. Cytological analysis showed that the rate of pollen mother cells (PMCs) with normal meiotic behavior in the IL5243 and IL5335 was to 89.93 and 90.22 %, respectively, and finally formed normal mature pollen. And meanwhile, the low frequency of abnormal chromosome behavior was observed in the IL5243 and IL5335, such as univalent, 8-shape bivalent, multivalent at diakinesis. At anaphase I, one or two lagging chromosomes were observed in some PMCs (3.95–5.15 %). The results of SSR marker analysis further confirmed that partial alien DNA of common wild rice has been transferred into the IL5243 and IL5335. These results implied that the IL5243 and IL5335 might be excellent bridging germplasm for exploring and utilizing the cold-tolerant gene of common wild rice. In addition, IL5243 and IL5335 would provide a better experimental system for understanding some epigenetic phenomenon induced by alien gene introgression.     

Fine mapping of a <Emphasis Type="Italic">Xantha</Emphasis> mutation in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

The recessive mutation of the XANTHA gene (XNT) transforms seedlings and plants into a yellow color, visually distinguishable from normal (green) rice. Thus, it has been introduced into male sterile lines as a distinct marker for rapidly testing and efficiently increasing varietal purity in seed and paddy production of hybrid rice. To identify closely linked markers and eventually isolate the XNT gene, two mapping populations were developed by crossing the xantha mutant line Huangyu B (indica) with two wild type japonica varieties; a total of 1,720 mutant type F₂ individuals were analyzed for fine mapping using polymorphic InDel markers and high dense microsatellite markers. The XNT gene was mapped on chromosome 11, within in a fragment of ~100 kb, where 13 genes are annotated. The NP_001067671.1 gene within the delimited region is likely to be a candidate XNT gene, since it encodes ATP-dependent chloroplast protease ATP-binding subunit clp A. However, no sequence differences were observed between the mutant and its parent. Bioinformatics analysis demonstrated that four chlorophyll deficient mutations that were previously mapped on the same chromosome are located outside the XNT region, indicating XNT is a new gene. The results provide useful DNA markers not only for marker assisted selection of the xantha trait but also its eventual cloning.     

Mapping of new resistance (<Emphasis Type="Italic">Vr2</Emphasis>, <Emphasis Type="Italic">Rm1</Emphasis>) and ornamental (<Emphasis Type="Italic">Di2</Emphasis>, <Emphasis Type="Italic">pl</Emphasis>) Mendelian trait loci in peach

Peach powdery mildew is one of the major diseases of the peach. Various sources of resistance to PPM have thus been identified, including the single dominant locus Vr2 carried by the peach rootstock ‘Pamirskij 5’. To map Vr2, a linkage map based on microsatellite markers was constructed from the F₂ progeny (WP²) derived from the cross ‘Weeping Flower Peach’ × ‘Pamirskij 5’. Self-pollinations of the parents were also performed. Under greenhouse conditions, all progenies were scored after artificial inoculations in two classes of reactions to PPM (resistant/susceptible). In addition to Vr2, WP² segregated for three other traits from ‘Weeping Flower Peach’: Rm1 for green peach aphid resistance, Di2 for double-flower and pl for weeping-growth habit. With their genomic locations unknown or underdocumented, all were phenotyped as Mendelian characters and mapped: Vr2 mapped at the top of LG8, at 3.3 cM, close to the CPSCT018 marker; Rm1 mapped at the bottom of LG1, at a position of 116.5 cM, cosegregating with the UDAp-467 marker and in the same region as Rm2 from ‘Rubira’^®; Di2 mapped at 28.8 cM on LG6, close to the MA027a marker; and pl mapped at 44.1 cM on LG3 between the MA039a and SSRLG3_16m46 markers. Furthermore, this study revealed, for the first time, a pseudo-linkage between two traits of the peach: Vr2 and the Gr locus, which controls the red/green color of foliage. The present work therefore constitutes a significant preliminary step for implementing marker-assisted selection for the four major traits targeted in this study.     

Application of the TRAP technique to lettuce (<Emphasis Type="Italic">Lactuca sativa</Emphasis> L<Emphasis Type="Italic">.</Emphasis>) genotyping

Summary To demonstrate the applicability of the target region amplification polymorphism (TRAP) marker technique to lettuce genotyping, we fingerprinted 53 lettuce (Lactuca sativa L.) cultivars and six wild accessions (three from each of the two wild species, L. saligna L. and L. serriola L.). Seven hundred and sixty-nine fragments from 50 to 900 bp in length were amplified in 10 PCR reactions using 10 fixed primers in combination with four fluorescent labeled arbitrary primers. Three hundred and eighty-eight of these fragments were polymorphic among the 59 Lactuca entries and 107 fragments were polymorphic among the 53 lettuce cultivars and the six wild accessions; 251 fragments were present only in the wild species. These markers not only discriminated all cultivars, but also revealed the evolutionary relationship among the three species: L. sativa, the cultivated species, is more closely related to L. serriola than to L. saligna. Cluster analysis grouped the cultivars by horticultural types with a few exceptions. These results are consistent with previous findings using RFLP, AFLP, and SAMPL markers. The TRAP markers revealed significant differences in genetic variability among horticultural types, measured by the average genetic similarity among the cultivars of the same type. Within the sample set, the leaf type and butterhead types possessed relatively high genetic variability, the iceberg types had moderate variability and the romaine types had the lowest variability. The genetic behavior of TRAP markers was assessed with a mapping population of 45 recombinant inbred lines (RILs) derived from an interspecific cross between L. serriola and L. sativa. Almost all the markers segregated in the expected 1:1 Mendelian ratio and are being incorporated into the existing lettuce linkage maps. Our results indicate that the TRAP markers can provide a powerful technique for fingerprinting lettuce cultivars. The U.S. Government's right to retain a non-exclusive, royalty-free license in and to any copyright is acknowledged.     

Genetic effects of nine <Emphasis Type="Italic">Gossypium barbadense</Emphasis> L. chromosome substitution lines in top crosses with five elite Upland cotton <Emphasis Type="Italic">G. hirsutum</Emphasis> L. cultivars

Crosses between Gossypium barbadense L and Gossypium hirsutum L. (Upland cotton) have produced limited success in introgressing fiber quality genes into the latter. Chromosome substitution lines (CSBL) have complete chromosomes or chromosome arms from G. barbadense, line 3-79, substituted for the corresponding chromosome or arms in G. hirsutum in a near isogenic background of TM-1. We top crossed nine CSBL and their parents (TM-1 and 3-79) with five cultivars. Parental lines and their F₂ populations were evaluated in four environments for agronomic and fiber quality traits. The CSBL and their F₂ hybrids showed wide ranges for both agronomic and fiber traits of economic importance. Genetic analysis showed that additive variances were larger than dominance variances for lint percentage, boll weight, lint yield, fiber length, strength, elongation, micronaire, and yellowness; whereas, dominance variances were larger than additive variances only for uniformity of fiber length and equal for fiber reflectance. For all traits, except boll weight and lint yield, significant additive effects of one or more chromosomes from 3-79 in TM-1 background were greater than the corresponding TM-1 chromosome. In addition, we identified specific chromosomes from G. barbadense (3-79) that carry alleles for improvements in specific fiber quality traits in Upland cotton. Favorable additive effects of individual chromosomes or chromosome segments from 3-79 relative to corresponding chromosomes or chromosomes segments from TM-1 were identified in this study as follows: Lint percentage, chromosome/arms 10, 16-15; longer fibers, chromosome/arms 01, 11sh, 26Lo; more uniform fibers, chromosomes/arms 01, 11sh, 10, 17-11; stronger fibers, chromosome/arms 01, 11sh, 12sh, 26Lo, 17-11; fiber elongation, chromosomes/arms 01, 11sh, 26Lo, 10, 17-11; reduced fiber micronaire, chromosome/arms 01, 12sh, 4-15, 16-15, 17-11; fibers with more reflectance, chromosome/arms 10, 4-15, 16-15, 17-11; fiber with less yellowness, chromosome arms 4-15, 17-11. Based on the present study, we concluded that by using CSBL, favorable fiber quality alleles can be introgressed into Upland cotton, thus greatly improving the breeder’s ability for improvement of Upland cotton for a variety of traits. These data should provide useful genetic information to the cotton breeding industry at large.     

Microsporogenesis of <Emphasis Type="Italic">Rps</Emphasis>8/<Emphasis Type="Italic">rps</Emphasis>8 heterozygous soybean lines

Phytophthora root and stem rot caused by Phytophthora sojae, is one of the most damaging diseases of soybean, for which management is principally done by planting resistant cultivars with race specific resistance which are conferred by Rps (Resistance to Phytophthora sojae) genes. The Rps8 locus, identified in the South Korean landrace PI 399073, is located in a 2.23 Mbp region on soybean chromosome 13. In eight cv. Williams (rps8/rps8) × PI 399073 (Rps8/Rps8) populations, this region exhibited strong segregation distortion. In a cross between the South Korean lines PI 399073 (Rps8/Rps8) and PI 408211B (multiple Rps genes) this region segregated in a Mendelian fashion. In this study, microsporogenesis was evaluated to identify meiotic abnormalities that may be associated with the segregation distortion of the Rps8 region. Pollen was collected from greenhouse-grown plants of the parental genotypes: Williams, PI 399073, and PI 408211B; as well as selected Rps8/rps8 RILs from Williams × PI 399073 BC₄F_2:3 and PI 399073 × PI 408211B F_4:5 populations. There were no differences for pollen viability among the genotypes. However, for PI 399073, a mix of dyads, triads, tetrads and pentads was observed. A high frequency of meiotic abnormalities including fragments, laggards, multinucleated microspores; and microcytes containing DNA was also observed in Rps8/rps8 Williams × PI 399073 BC₄F_2:3 RILs. These meiotic abnormalities may contribute to the high degree of segregation distortion present in the Williams × PI 399073 populations.     

Identification of quantitative trait loci involved in resistance to <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">syringae</Emphasis> in pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.)

Pseudomonas syringae is the main pathogen responsible for bacterial blight disease in pea and can cause yield losses of 70%. P. syringae pv. pisi is prevalent in most countries but the importance of P. syringae pv. syringae (Psy) is increasing. Several sources of resistance to Psy have been identified but genetics of the resistance is unknown. In this study the inheritance of resistance to Psy was studied in the pea recombinant inbred line population P665 × ‘Messire’. Results suggest a polygenic control of the resistance and two quantitative trait loci (QTL) associated with resistance, Psy1 and Psy2, were identified. The QTL explained individually 22.2 and 8.6% of the phenotypic variation, respectively. In addition 21 SSR markers were included in the P665 × ‘Messire’ map, of which six had not been mapped on the pea genome in previous studies.     

Increased resistance to <Emphasis Type="Italic">Ustilago zeae</Emphasis> and <Emphasis Type="Italic">Fusarium verticilliodes</Emphasis> in maize inbred lines bred for <Emphasis Type="Italic">Fusarium graminearum</Emphasis> resistance

Preliminary field observations in our maize breeding nurseries indicated that breeding for improved resistance to gibberella ear rot (Fusarium graminearum) in maize may indirectly select for resistance to another ear disease, common smut (Ustilago zeae). To investigate this, we compared the disease severity ratings obtained on 189 maize inbreds, eight of which included our inbreds developed with selection for gibberella ear rot resistance after field inoculation and breeding for 8–10 years. No correlation was found between disease severities for the 189 inbreds but the eight gibberella-resistant lines were consistently more resistant to smut. To further examine this relationship and to determine if these eight inbreds would be useful for developing inbreds with either common smut or fusarium ear rot (F. verticilliodes) resistance, we conducted a Griffing’s diallel analysis on six inbreds of maize, four with high levels of gibberella ear rot resistance representing all of the pedigree groups in our eight gibberella lines, and two with very low levels. Our most gibberella ear rot resistant inbreds, CO433 and CO441, had the lowest disease ratings for all three diseases, the consistently largest general combining ability effects and several significant specific combining ability effects. It was concluded that some inbreds bred specifically for gibberella ear rot would also be useful in breeding for resistance to common smut and fusarium ear rot.     

The inheritance of chemical phenotype in <Emphasis Type="Italic">Cannabis</Emphasis><Emphasis Type="Italic">sativa</Emphasis> L. (IV): cannabinoid-free plants

A genetic factor that blocks the cannabinoid biosynthesis in Cannabis sativa has been investigated. Crosses between cannabinoid-free material and high content, pharmaceutical clones were performed. F₁s were uniform and had cannabinoid contents much lower than the mean parental value. Inbred F₂ progenies segregated into discrete groups: a cannabinoid-free chemotype, a chemotype with relatively low cannabinoid content and one with relatively high content, in a monogenic 1:2:1 ratio. In our model the cannabinoid knockout factor is indicated as a recessive allele o, situated at locus O, which segregates independently from previously presented chemotype loci. The genotype o/o underlies the cannabinoid-free chemotype, O/o is expressed as an intermediate, low content chemotype, and O/O is the genotype of the high content chemotype. The data suggests that locus O governs a reaction in the pathway towards the phenolic cannabinoid precursors. The composition of terpenoids and various other compound classes of cannabinoid-free segregants remains unaffected. Backcrossing produced cannabinoid-free homologues of pharmaceutical production clones with potential applications in pharmacological research. A new variant of the previously presented allele ‘B ₀’, that almost completely obstructs the conversion of CBG into CBD, was also selected from the source population of the cannabinoid knockout factor.     

A simple,rapid and reliable bioassay for evaluating seedling vigor under submergence in <Emphasis Type="Italic">indica</Emphasis> and <Emphasis Type="Italic">japonica</Emphasis> rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Submergence is a major stress causing yield losses particularly in the direct-seeded rice cultivation system and necessitates the development of a simple, rapid and reliable bioassay for a large scale screening of rice germplasms with tolerance against submergence stress. We developed two new bioassay methods that were based primarily on the seedling vigor evaluated by the ability of fast shoot elongation under submerged conditions, and compared their effectiveness with two other available methods. All four bioassay methods using cultivars of 7 indica and 6 japonica types revealed significant and consistent cultivar differences in seedling vigor under submergence and/or submergence tolerance. Japonica cultivars were more vigorous than indica cultivars, with Nipponbare being the most vigorous. The simplest test tube method showed the highest correlations to all other methods. Our results suggest that seedling vigor serves as a submergence avoidance mechanism and confers tolerance on rice seedlings to flooding during early crop establishment. A possible relationship is discussed between seedling vigor based on fast shoot elongation and submergence tolerance defined by recovery from submergence stress.     

Development of microsatellite markers in cultivated and wild species of sections <Emphasis Type="Italic">Cepa</Emphasis> and <Emphasis Type="Italic">Phyllodolon</Emphasis> in <Emphasis Type="Italic">Allium</Emphasis>

The potential of microsatellite markers for use in genetic studies has been evaluated in Allium cultivated species (Allium cepa, A. fistulosum) and its allied species (A. altaicum, A. galanthum, A. roylei, A. vavilovii). A total of 77 polymerase chain reaction (PCR) primer pairs were employed, 76 of which amplified a single product or several products in either of the species. The 29 AMS primer pairs derived from A. cepa and 46 microsatellites primer pairs from A. fistulosum revealed a lot of polymorphic amplicons between seven Allium species. Some of the microsatellite markers were effective not only for identifying an intraspecific F₁ hybrid between shallot and bulb onion but also for applying to segregation analyses in its F₂ population. All of the microsatellite markers can be used for interspecific taxonomic analyses among two cultivated and four wild species of sections Cepa and Phyllodolon in Allium. Generally, our data support the results obtained from recently performed analyses using molecular and morphological markers. However, the phylogeny of A. roylei, a threatened species with several favorable genes, was still ambiguous due to its different positions in each dendrogram generated from the two primer sets originated from A. cepa and A. fistulosum.     

Resistance screening of <Emphasis Type="Italic">Coffea</Emphasis> spp. accessions for <Emphasis Type="Italic">Pratylenchus coffeae</Emphasis> and <Emphasis Type="Italic">Radopholus arabocoffeae</Emphasis> in Vietnam

Coffee varieties with resistance for the plant-parasitic nematodes Pratylenchus coffeae and Radopholus arabocoffeae are limited in Vietnam. A selection of imported varieties and high yield varieties of Arabica coffee in Vietnam were evaluated for resistance to both plant-parasitic nematode species in Northern Vietnam. The same experiments were carried out with hybrid arabica coffee, three selected clones of Coffea canephora and one clone of Coffea excelsa in the Western Highland of Vietnam. The screened coffee accessions from Ethiopia (KH1, KH13, KH20, KH21, KH29, and KH31) were susceptible and good host for P. coffeae. Also accessions 90P4 (Portugal) and Oro azteca (Mexico) had a reproduction factor Rf > 1. Pluma Hidalgo (Mexico), 90/6 (Vietnam), 90P3 (Portugal), 90P2 (Vietnam), Variedad (Mexico), 90T (Portugal), and Garnica (Mexico) were poor hosts (Rf < 1) but not tolerant to P. coffeae, expressed by a reduction of root weight compared to untreated control plants. Most of the coffee accessions tested in Northern Vietnam were intolerant to R. arabocoffeae, except 90T which showed no reduction of root weight, even at high initial nematode densities (4,000/pot). Good hosts for R. arabocoffeae were Variedad, KH1, KH21, KH29, KH20, KH31, and KH13 with Rf > 1. Pluma Hidalgo, 90/6, 90P3, 90P2, 90T, Oro azteca, and Garnica were poor hosts (Rf < 1). In the Western Highland experiment, all arabica coffee accessions were susceptible for P. coffeae with Rf ranging from 1.41 to 1.59. Tolerance to P. coffeae was found in C. liberica var. Dewevrei, Hong34 and Nhuantren. Coffea excelsa, Hong34, Nhuantren, and H1C19 were tolerant to R. arabocoffeae at the highest inoculation density (4,000 nematodes/pot). The most susceptible accessions were Nhuantren and K55. Resistance (Rf < 1) to R. arabocoffeae was found in C. liberica var. Dewevrei and Hong34. This article reports on the first screening for resistance and tolerance to P. coffeae and R. arabocoffeae in coffee accessions in Vietnam and shows promising results for enhanced coffee-breeding.     

<Emphasis Type="Italic">Agrobacterium</Emphasis> mediated transformation of indica rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) for insect resistance

The rice leaffolder (RLF), Cnaphalocrocis medinalis is an important pest of rice that causes severe damage in many areas of the world. The plants were transformed with fully modified (plant codon optimized) synthetic Cry1C coding sequences as well as with the hpt and gus genes, coding for hygromycin phosphotransferase and β-glucuronidase, respectively. Cry1C sequences placed under the control of doubled 35S promoter plus the AMV leader sequence, and hpt and gus genes driven by cauliflower mosaic virus 35S promoter, were used in this study. Embryogenic calli after cocultivation with Agrobacterium were selected on the medium containing hygromycin B. A total of 67 hygromycin-resistant plants were regenerated. PCR and Southern blot analyses of primary transformants revealed the stable integration of Cry1C coding sequences into the rice genome with predominant single copy integration. R₁ progeny plants disclosed a monogenic pattern (3:1) of transgene segregation as confirmed by molecular analyses. These transgenic lines were highly resistant to rice leaffolder (RLF), Cnaphalocrocis medinalis as revealed by insect bioassay.