Teresa Millán

Draft genome sequence of chickpea (Cicer arietinum) provides a resource for trait improvement

Chickpea (Cicer arietinum) is the second most widely grown legume crop after soybean, accounting for a substantial proportion of human dietary nitrogen intake and playing a crucial role in food security in developing countries. We report the ∼738-Mb draft whole genome shotgun sequence of CDC Frontier, a kabuli chickpea variety, which contains an estimated 28,269 genes. Resequencing and analysis of 90 cultivated and wild genotypes from ten countries identifies targets of both breeding-associated genetic sweeps and breeding-associated balancing selection. Candidate genes for disease resistance and agronomic traits are highlighted, including traits that distinguish the two main market classes of cultivated chickpea—desi and kabuli. These data comprise a resource for chickpea improvement through molecular breeding and provide insights into both genome diversity and domestication.

Phylogenetic analysis in the genus Cicer and cultivated chickpea using RAPD and ISSR markers

Abstract Seventy five accessions belonging to 14 species of the genus Cicer were analysed with PCR-based molecular markers to determine their phylogenetic relationships. Eight of the species were annuals and included the Section Monocicer which contains cultivated chickpea (Cicer arietinum L.). The remaining six species were perennials (five from Section Polycicer and one from Section Acanthocicer). More than one accession per species was analysed in most of the wild species; within C. arietinum, 26 accessionsincluding Kabuli and Desi types, were studied. RAPD analyses using 12 primers gave 234 polymorphic fragments. Variability within species was detected. A dendrogram based on the Jaccard similarity index showed that the distribution pattern of variability between species was related to both growth habit and geographical origin. An accession of Cicer reticulatum was closer to accessions of Cicer echinospermum than to the four remaining of C. reticulatum, suggesting the possibility of gene flow between species. Cluster analysis for cultivated chickpea differentiated Kabuli and Desi types but we did not detect a clear relationship between groups and the geographical origin of the accessions.

Chickpea molecular breeding: New tools and concepts

SummaryChickpea is a cool season grain legume of exceptionally high nutritive value and most versatile food use. It is mostly grown under rain fed conditions in arid and semi-arid areas around the world. Despite growing demand and high yield potential, chickpea yield is unstable and productivity is stagnant at unacceptably low levels. Major yield increases could be achieved by development and use of cultivars that resist/tolerate abiotic and biotic stresses. In recent years the wide use of early maturing cultivars that escape drought stress led to significant increases in chickpea productivity. In the Mediterranean region, yield could be increased by shifting the sowing date from spring to winter. However, this is hampered by the sensitivity of the crop to low temperatures and the fungal pathogen Ascochyta rabiei. Drought, pod borer (Helicoverpa spp.) and the fungus Fusarium oxysporum additionally reduce harvests there and in other parts of the world. Tolerance to rising salinity will be a future advantage in many regions. Therefore, chickpea breeding focuses on increasing yield by pyramiding genes for resistance/tolerance to the fungi, to pod borer, salinity, cold and drought into elite germplasm. Progress in breeding necessitates a better understanding of the genetics underlying these traits. Marker-assisted selection (MAS) would allow a better targeting of the desired genes. Genetic mapping in chickpea, for a long time hampered by the little variability in chickpea’s genome, is today facilitated by highly polymorphic, co-dominant microsatellite-based markers. Their application for the genetic mapping of traits led to inter-laboratory comparable maps. This paper reviews the current situation of chickpea genome mapping, tagging of genes for ascochyta blight, fusarium wilt resistance and other traits, and requirements for MAS. Conventional breeding strategies to tolerate/avoid drought and chilling effects at flowering time, essential for changing from spring to winter sowing, are described. Recent approaches and future prospects for functional genomics of chickpea are discussed.

Using RAPDs to study phylogenetic relationships in Rosa.

Nineteen species of rose (Rosa sp.) were analysed using Random Amplified Polymorphic DNA markers (RAPD). Each 10-base-long arbitrary primer produced a specific DNA banding pattern that grouped plants belonging to the same species and botanical sections as predicted from their genetic background. One hundred and seventy-five amplification products were examined by cluster analysis to assess the genetic relationships among species and their genetic distances. All of the accessions belonging to 1 species grouped together before branching to other species. Dendrograms constructed for intra- and inter-specific studies showed a good correlation with previous classifications by different authors based on morphological and cariological studies. Our results show that the RAPD technique is a sensitive and precise tool for genomic analysis in rose, being useful in assigning unclassified accessions to specific taxonomic groups or else allowing accessions classified by traditional criteria to be re-classified.

A linkage map of chickpea (Cicer arietinum L.) based on populations from Kabuli × Desi crosses: location of genes for resistance to fusarium wilt race 0

Two recombinant inbred line (RIL) populations derived from intraspecific crosses with a common parental line (JG62) were employed to develop a chickpea genetic map. Molecular markers, flower colour, double podding, seed coat thickness and resistance to fusarium wilt race 0 (FOC-0) were included in the study. Joint segregation analysis involved a total of 160 markers and 159 RILs. Ten linkage groups (LGs) were obtained that included morphological markers and 134 molecular markers (3 ISSRs, 13 STMSs and 118 RAPDs). Flower colour (B/b) and seed coat thickness (Tt/tt) appeared to be linked to STMS (GAA47). The single-/double-podding locus was located on LG9 jointly with two RAPD markers and STMS TA80. LG3 included a gene for resistance to FOC-0 (Foc01/foc01) flanked by RAPD marker OPJ20600 and STMS marker TR59. The association of this LG with FOC-0 resistance was confirmed by QTL analysis in the CA2139 × JG62 RIL population where two genes were involved in the resistance reaction. The STMS markers enabled comparison of LGs with preceding maps.

A new QTL for Ascochyta blight resistance in an RIL population derived from an interspecific cross in chickpea

SummaryA linkage map in a population of recombinant inbred lines (RILs) derived from an interspecific cross between Cicer arietinum (ILC72) × Cicer reticulatum (Cr5-10), resistant and susceptible to blight, caused by Ascochyta rabiei, respectively, was obtained using RAPD, ISSR, STMS, isozyme (Pdf6) and flower colour (pink/white) markers. The map comprised ten linkage groups and covered a distance of 601.2 cM. When the population was evaluated for reaction to Ascochyta blight under field conditions by determining the Area Under the Disease Progress Curve (AUDPC), the distribution of frequencies was bimodal: most of the lines had an intermediate reaction, fewer were nearly as susceptible as the susceptible parent and none had values close to the resistant parent. A QTL explaining 28% of the variation in resistance was located in linkage group 2 (LG2). Five RAPD markers on this linkage group showed significant association with resistance (OPX04372, UBC881621, OPAI09746, OPAI09352 and OPAC12700) and the major QTL peak lay midway between OPAI09746 and UBC881621 which are 14.1 cM apart. Contrary to other studies, no association of linkage group 4 with resistance was found. The QTL for resistance to Ascochyta blight in this study is therefore different from QTLs for this character reported in other interspecific crosses and may be the same as that reported in linkage group 2 in intraspecific crosses where genes for resistance to races of Fusarium oxysporum f. sp. ciceri, causing wilt, are also located.

A chromosomal genomics approach to assess and validate the desi and kabuli draft chickpea genome assemblies

With the expansion of next-generation sequencing technology and advanced bioinformatics, there has been a rapid growth of genome sequencing projects. However, while this technology enables the rapid and cost-effective assembly of draft genomes, the quality of these assemblies usually falls short of gold standard genome assemblies produced using the more traditional BAC by BAC and Sanger sequencing approaches. Assembly validation is often performed by the physical anchoring of genetically mapped markers, but this is prone to errors and the resolution is usually low, especially towards centromeric regions where recombination is limited. New approaches are required to validate reference genome assemblies. The ability to isolate individual chromosomes combined with next-generation sequencing permits the validation of genome assemblies at the chromosome level. We demonstrate this approach by the assessment of the recently published chickpea kabuli and desi genomes. While previous genetic analysis suggests that these genomes should be very similar, a comparison of their chromosome sizes and published assemblies highlights significant differences. Our chromosomal genomics analysis highlights short defined regions that appear to have been misassembled in the kabuli genome and identifies large-scale misassembly in the draft desi genome. The integration of chromosomal genomics tools within genome sequencing projects has the potential to significantly improve the construction and validation of genome assemblies. The approach could be applied both for new genome assemblies as well as published assemblies, and complements currently applied genome assembly strategies.

Tagging and mapping a second resistance gene for Fusarium wilt race 0 in chickpea

A second gene conferring resistance to the chickpea wilt pathogen, Fusarium oxysporum f. sp ciceris race 0, has been mapped to linkage group 2 (LG2) of the chickpea genetic map. Resistance to race 0 is controlled by two genes which segregate independently; one present in accession JG62 (Foc01/foc01) and mapping to LG5 and the second present in accession CA2139 (Foc02/foc02) but remaining unmapped. Both genes separately confer complete resistance to race 0 of the wilt pathogen. Using a Recombinant Inbred Line (RIL) population that segregated for both genes (CA2139 × JG62) and the genotypic information provided by two markers flanking Foc01/foc01 ten resistant lines containing the resistant allele Foc02/foc02 were selected. Genotypic analysis using these ten resistant lines paired with ten susceptible RILs, selected in the same population, revealed that sequence tagged microsatellite sites (STMS) markers sited on LG2 were strongly associated with Foc02/foc02. Linkage analysis, using data from two mapping populations (CA2139/JG62 and CA2156/JG62), located Foc02/foc02 in a region where genes for resistance to wilt races 1, 2, 3, 4 and 5 have previously been reported and which is highly saturated with tightly-linked STMS markers that could be used in marker-assisted selection (MAS).

Integration of new CAPS and dCAPS-RGA markers into a composite chickpea genetic map and their association with disease resistance

A composite linkage map was constructed based on two interspecific recombinant inbred line populations derived from crosses between Cicer arietinum (ILC72 and ICCL81001) and Cicer reticulatum (Cr5-10 or Cr5-9). These mapping populations segregate for resistance to ascochyta blight (caused by Ascochyta rabiei), fusarium wilt (caused by Fusarium oxysporum f. sp. ciceris) and rust (caused by Uromyces ciceris-arietini). The presence of single nucleotide polymorphisms in ten resistance gene analogs (RGAs) previously isolated and characterized was exploited. Six out of the ten RGAs were novel sequences. In addition, classes RGA05, RGA06, RGA07, RGA08, RGA09 and RGA10 were considerate putatively functional since they matched with several legume expressed sequences tags (ESTs) obtained under infection conditions. Seven RGA PCR-based markers (5 CAPS and 2 dCAPS) were developed and successfully genotyped in the two progenies. Six of them have been mapped in different linkage groups where major quantitative trait loci conferring resistance to ascochyta blight and fusarium wilt have been reported. Genomic locations of RGAs were compared with those of known Cicer R-genes and previously mapped RGAs. Association was detected between RGA05 and genes controlling resistance to fusarium wilt caused by races 0 and 5.

Development of chickpea near-isogenic lines for fusarium wilt

Four pairs of near-isogenic lines (NILs) of chickpea with resistance/susceptibility to Fusarium oxysporum f. sp. ciceris (Foc) have been developed in this study. These lines were produced by searching in advanced recombinant inbred lines (RILs) that are segregating for Foc race 5 based on a phenotypic screening. The sequence tagged microsatellite (STMS) marker TA59, closely linked to wilt resistance genes on linkage group 2 (LG2) of the chickpea map, was used to assist the selection of resistant or susceptible genotypes. The NILs were also characterized for disease reaction to Foc races 1A, 2, 3 and 4. Resistance, susceptibility and slow wilting reactions were found in these NILs. Our results suggest that more than one gene controls the resistance to race 5. Combination of the major gene foc-5 linked to TA59 with other gene/s appears to be required to complete resistance, and the absence of these unknown genes leads to slow wilting reactions. The independent differential responses to races 2 and 3 observed in three NILs could be explained as recombination events. This result suggests that foc-2 and foc-3 are delimiting points at opposite ends of a genomic region that includes the remaining foc genes and the TA59 marker. This set of NILs has great potential for studying the genetics and mechanisms of wilt resistance. In addition, the NIL RIP8-94-11 can be used as differential line for Foc race 3; it showed a clear resistance reaction to race 3 and susceptibility to the other Foc races.