Alain Ghesquière

Aroma in rice: genetic analysis of a quantitative trait

A new approach was developed which succeeded in tagging for the first time a major gene and two QTLs controlling grain aroma in rice. It involved a combination of two techniques, quantification of volatile compounds in the cooking water by gas chromatography, and molecular marker mapping. Four types of molecular marker were used (RFLPs, RAPDs, STSs, isozymes). Evaluation and mapping were performed on a doubled haploid line population which (1) conferred a precise character evaluation by enabling the analysis of large quantities of grains per genotype and (2) made possible the comparison of gas chromatography results and sensitive tests. The population size (135 lines) provided a good mapping precision. Several markers on chromosome 8 were found to be closely linked to a major gene controlling the presence of 2-acetyl-1-pyrroline (AcPy), the main compound of rice aroma. Moreover, our results showed that AcPy concentration in plants is regulated by at least two chromosomal regions. Estimations of recombination fractions on chromosome 8 were corrected for strong segregation distortion. This study confirms that AcPy is the major component of aroma. Use of the markers linked to AcPy major gene and QTLs for marker-assisted selection by successive backcrosses may be envisaged.

Heredity and genetic mapping of domestication-related traits in a temperate japonica weedy rice

Abstract Rice is often found as various weedy forms in temperate or newly cultivated rice growing regions throughout the world. The emergence of these forms in the absence of true wild rice remains unclear. A genetic analysis of domestication-related traits (weed syndrome) has been conducted to better understand the appearance of these plants in rice fields. A doubled haploid (DH) population was derived from a cross between a japonica variety and a weedy plant collected in Camargue (France) to set up a genetic linkage map consisting of 68 SSR and 31 AFLP loci. Five qualitative traits related to pigmentation of different organs and 15 developmental and morphological quantitative traits were scored for genes and QTLs mapping. Despite a good reactivity in anther culture and a high fertility of the DH lines, segregation distortions were observed on chromosomal segments bearing gametophytic and sterility genes and corresponded to various QTLs evidenced in indica×japonica distant crosses. Mapping of the coloration genes was found to be in agreement with the presence of several genes previously identified and according to the genetic model governing the synthesis and distribution of anthocyan pigment in the plant. In addition, the main specific traits of weedy forms revealed the same genes/QTLs as progeny derived from a cross between Oryza sativa and its wild progenitor O. rufipogon. A large variation for most characters was found in the DH population, including transgressive variation. Significant correlations were observed between morphology and traits related to weeds and corresponded to a distinct colocalization of most of the QTLs on a limited number of chromosomal regions. The significance of these results on the origin of weedy forms and the de-domestication process is discussed.

A first interspecific Oryza sativa × Oryza glaberrima microsatellite-based genetic linkage map.

Abstract Oryza glaberrima is an endemic African cultivated rice species. To provide a tool for evaluation and utilisation of the potential of O. glaberrima in rice breeding, we developed an interspecific O. glaberrima×Oryza sativa genetic linkage map. It was based on PCR markers, essentially microsatellites and STSs. Segregation of markers was examined in a backcross (O. sativa/O. glaberrima//O. sativa) population. Several traits were measured on the BC1 plants, and major genes and QTLs were mapped for these traits. Several of these genes correspond well to previously identified loci. The overall map length was comparable to those observed in indica×japonica crosses, indicating that recombination between the two species occurs without limitation. However, three chromosomes show discrepancies with the indica×japonica maps. The colinearity with intraspecific maps was very good, confirming previous cytological observations. A strong segregation-distortion hot spot was observed on chromosome 6 near the waxy gene, indicating the presence of s10, a sporo-gametophytic sterility gene previously identified by Sano (1990). The main interests of such a PCR-based map for African rice breeding are discussed, including gene and QTL localisation, marker-assisted selection, and the development of interspecific introgression lines.

The Genetic Basis of High Resistance to Rice Yellow Mottle Virus (RYMV) in Cultivars of Two Cultivated Rice Species

Three cultivars of Oryza sativa (IR64, Azucena, and Gigante) and four cultivars of O. glaberrima (Tog5681, Tog5673, CG14, and SG329) were evaluated for their resistance to two isolates of rice yellow mottle virus (RYMV) by enzyme-linked immunosorbent assay (ELISA) and symptomatology. Cultivars Tog5681 and Gigante were highly resistant, and no symptoms were observed when either virus isolate was inoculated at 10 or 20 days postgermination and assayed by ELISA at 7, 14, 22, 35, 50, or 64 days postinoculation. Azucena showed a partial resistance, whereas the other cultivars were susceptible. Symptom appearance was associated with increase in ELISA absorbance in the systemically infected leaves. The best discrimination among the cultivars occurred when the plants were inoculated at 10 days postgermination. Crosses were made between the highly resistant (Gigante and Tog5681) and the susceptible (IR64) cultivars to determine the genetic basis of resistance to RYMV. Evaluation of F1 hybrids and interspecific progenies, as well as the segregation of resistance in F2 and F3 lines of the IR64 × Gigante cross, provided results consistent with the presence of a single recessive resistance gene common to Tog5681 and Gigante.

Genetic basis and mapping of the resistance to rice yellow mottle virus. I. QTLs identification and relationship between resistance and plant morphology

Abstract Rice yellow mottle virus (RYMV) resistance QTLs were mapped in a doubled-haploid population of rice, ‘IR64/Azucena’. Disease impact on plant morphology and development, expression of symptoms and virus content were evaluated in field conditions. Virus content was also assessed in a growth chamber. RYMV resistance was found to be under a polygenic determinism, and 15 QTLs were detected on seven chromosomal fragments. For all of the resistance QTLs detected, the favourable allele was provided by the resistant parent ‘Azucena’. Three regions were determined using different resistance parameters and in two environments. On chromosome 12, a QTL of resistance that had already been detected in this population and another indica/japonica population was confirmed both in the field and under controlled conditions. Significant correlations were observed between resistance and tillering ability, as measured on control non-inoculated plants. In addition, the three genomic fragments involved in resistance were also involved in plant architecture and development. In particular, the semi-dwarfing gene sd-1, on chromosome 1, provided by the susceptible parent, ‘IR64’, mapped in a region where resistance QTLs were detected with most of the resistance parameters. In contrast, the QTL of resistance mapped on chromosome 12 was found to be independent of plant morphology.

Rice genomics: Present and future

A review of the present and future of rice genomics is presented. Rice is a model species for cereals as well as a very important crop. Its genome has been the focus of many mapping experiments associated with QTL localization. These genetic maps now serve as a background for physical mapping, genome sequencing and gene discovery. Recent progress are reviewed. The next step in rice genomics is functional genomics with the determination of the function of the genes. The most straightforward approaches are discussed.

Genetic basis and mapping of the resistance to Rice yellow mottle virus. III. Analysis of QTL efficiency in introgressed progenies confirmed the hypothesis of complementary epistasis between two resistance QTLs

Abstract  Our previous studies have hypothesised that a complementary epistasis between a QTL located on chromosome 12 and a QTL located on chromosome 7 was one of the major genetic factors controlling partial resistance to Rice yellow mottle virus (RYMV). We report research undertaken to verify this hypothesis and to introgress the resistant allele of these two QTLs from an upland resistant japonica variety, Azucena, into a lowland susceptible indica variety IR64. Three cycles of molecular marker-assisted back cross breeding were performed using RFLP and microsatellite markers. Resistance to RYMV was evaluated in F2 and F3 offspring of the BC1 and BC2 generations. Marker-assisted introgression (MAI) was very efficient: in the selected BC3 progeny the proportion of the recipient genome was close to 95% for the ten non-carrier chromosomes, and the length of the donor chromosome segment surrounding the two QTLs was less than 20 cM. The relevancy of the complementary epistasis genetic model proposed previously was confirmed experimentally: in BC1 and BC2 generations only F3 lines having the allele of the resistant parent on QTL12 and QTL7 show partial resistance to RYMV. Comparison of our experimental process of MAI with the recommendations of analytic and simulation studies pointed out the methodological flexibility of MAI. Our results also confirmed the widely admitted, but rarely verified, assumption that QTL-alleles detected in segregating populations could be treated as units of Mendelian inheritance and that the incorporation of these alleles into elite lines would result in an enhanced performance. The next step will be the design of tools for the routine use of molecular markers in breeding for partial resistance to RYMV and the development of material for the analysis of resistance mechanisms and the structure of a virus resistance gene in rice.

Fine genetic mapping of a gene required for Rice yellow mottle virus cell-to-cell movement

Abstract. The very high resistance to Rice yellow mottle virus observed in the two rice varieties Gigante (Oryza sativa) and Tog 5681 (O. glaberrima) is monogenic and recessive. Bulked segregant analysis was carried out to identify AFLP markers linked to the resistance gene. Mapping of PCR-specific markers, CAPS and microsatellite markers on 429 individuals of an IR64 × Gigante F2 population pinpointed this resistance gene on the long arm of chromosome 4 in a 3.7-cM interval spanned by PCR markers. These markers also flanked the resistance gene of the O. glaberrima accession Tog 5681 and confirmed previous allelism tests. The rarity of this recessive natural resistance was in line with a resistance mechanism model based on point mutations of a host component required for cell-to-cell movement of the virus. Preliminary data on the genetic divergence between the two cultivated rice species in the vicinity of the resistance locus suggested that two different resistance alleles are present in Gigante and Tog 5681. A large set of recombinants is now available to envisage physical mapping and cloning of the gene.

Oryza Tag Line, a phenotypic mutant database for the Génoplante rice insertion line library

To organize data resulting from the phenotypic characterization of a library of 30 000 T-DNA enhancer trap (ET) insertion lines of rice (Oryza sativa L cv. Nipponbare), we developed the Oryza Tag Line (OTL) database (http://urgi.versailles.inra.fr/OryzaTagLine/). OTL structure facilitates forward genetic search for specific phenotypes, putatively resulting from gene disruption, and/or for GUSA or GFP reporter gene expression patterns, reflecting ET-mediated endogenous gene detection. In the latest version, OTL gathers the detailed morpho-physiological alterations observed during field evaluation and specific screens in a first set of 13 928 lines. Detection of GUS or GFP activity in specific organ/tissues in a subset of the library is also provided. Search in OTL can be achieved through trait ontology category, organ and/or developmental stage, keywords, expression of reporter gene in specific organ/tissue as well as line identification number. OTL now contains the description of 9721 mutant phenotypic traits observed in 2636 lines and 1234 GUS or GFP expression patterns. Each insertion line is documented through a generic passport data including production records, seed stocks and FST information. 8004 and 6101 of the 13 928 lines are characterized by at least one T-DNA and one Tos17 FST, respectively that OTL links to the rice genome browser OryGenesDB.

Large-scale characterization of Tos17 insertion sites in a rice T-DNA mutant library

We characterized the insertion sites of newly transposed copies of the tissue-culture-induced ty1-copia retrotransposon Tos17 in the Oryza Tag Line (OTL) T-DNA mutant library of rice cv. Nipponbare. While Nipponbare contains two native copies of Tos17 the number of additional copies, deduced from Southern blot analyses in a subset of 384 T-DNA lines and using a reverse transcriptase probe specific to the element, ranged from 1 to 8 and averaged 3.37. These copies were shown to be stably inherited and to segregate independently in the progenies of insertion lines. We took advantage of the absence of EcoRV restriction sites in the immediate vicinity of the 3′ LTR of the native copies of Tos17 in the genome sequence of cv. Nipponbare, thereby preventing amplification of corresponding PCR fragments, to efficiently and selectively amplify and sequence flanking regions of newly transposed Tos17 inserts. From 25,286 T-DNA plants, we recovered 19,252 PCR products (76.1%), which were sequenced yielding 14,513 FSTs anchored on the rice pseudomolecules. Following elimination of redundant sequences due to the presence of T-DNA plants deriving from the same cell lineage, these FSTs corresponded to 11,689 unique insertion sites. These unique insertions exhibited higher densities in subtelomeric regions of the chromosomes and hot spots for integration, following a distribution that remarkably paralleled that of Tos17 sites in the National Institute for Agrobiological Sciences (NIAS) library. The insertion sites were mostly found in genic regions (77.5%) and preferably in coding sequences (68.8%) compared to unique T-DNA insertion sites in the same materials (49.1% and 28.3%, respectively). Predicted non- transposable element (TE) genes prone to a high frequency of Tos17 integration (i.e. from 5 to 121 inserts) in the OTL T-DNA collection were generally found to be also hot spots for integration in the NIAS library. The 9,060 Tos17 inserts inserted into non TE genes were found to disrupt a total of 2,773 genes with an average of 3.27 inserts per gene, similar to that in the NIAS library (3.28 inserts per gene on average) whereas the 4,472 T-DNA inserted into genes in the same materials disrupted a total of 3,911 genes (1.14 inserts per gene on average). Interestingly, genes disrupted by both Tos17 and T-DNA inserts in the library represented only 14.9% and 10.6% of the complement of genes interrupted by Tos17 and T-DNA inserts respectively while 52.1% of the genes tagged by Tos17 inserts in the OTL library were found to be tagged also in the NIAS Tos17 library. We concluded that the first advantage in characterizing Tos17 inserts in a rice T-DNA collection lies in a complementary tagging of novel genes and secondarily in finding other alleles in a same genetic background, thereby greatly enhancing the library genome coverage and its overall value for implementing forward and reverse genetics strategies.