Genome-wide insertion–deletion (InDel) marker discovery and genotyping for genomics-assisted breeding applications in chickpea

We developed 21,499 genome-wide insertion–deletion (InDel) markers (2- to 54-bp in silico fragment length polymorphism) by comparing the genomic sequences of four (desi, kabuli and wild C. reticulatum) chickpea [Cicer arietinum (L.)] accessions. InDel markers showing 2- to 6-bp fragment length polymorphism among accessions were abundant (76.8%) in the chickpea genome. The physically mapped 7,643 and 13,856 markers on eight chromosomes and unanchored scaffolds, respectively, were structurally and functionally annotated. The 4,506 coding (23% large-effect frameshift mutations) and regulatory InDel markers were identified from 3,228 genes (representing 11.7% of total 27,571 desi genes), suggesting their functional relevance for trait association/genetic mapping. High amplification (97%) and intra-specific polymorphic (60–83%) potential and wider genetic diversity (15–89%) were detected by genome-wide 6,254 InDel markers among desi, kabuli and wild accessions using even a simpler cost-effective agarose gel-based assay. This signifies added advantages of this user-friendly genetic marker system for manifold large-scale genotyping applications in laboratories with limited infrastructure and resources. Utilizing 6,254 InDel markers-based high-density (inter-marker distance: 0.212 cM) inter-specific genetic linkage map (ICC 4958 × ICC 17160) of chickpea as a reference, three major genomic regions harboring six flowering and maturity time robust QTLs (16.4–27.5% phenotypic variation explained, 8.1–11.5 logarithm of odds) were identified. Integration of genetic and physical maps at these target QTL intervals mapped on three chromosomes delineated five InDel markers-containing candidate genes tightly linked to the QTLs governing flowering and maturity time in chickpea. Taken together, our study demonstrated the practical utility of developing and high-throughput genotyping of such beneficial InDel markers at a genome-wide scale to expedite genomics-assisted breeding applications in chickpea.     

Deploying QTL-seq for rapid delineation of a potential candidate gene underlying major trait-associated QTL in chickpea

A rapid high-resolution genome-wide strategy for molecular mapping of major QTL(s)/gene(s) regulating important agronomic traits is vital for in-depth dissection of complex quantitative traits and genetic enhancement in chickpea. The present study for the first time employed a NGS-based whole-genome QTL-seq strategy to identify one major genomic region harbouring a robust 100-seed weight QTL using an intra-specific 221 chickpea mapping population (desi cv. ICC 7184 × desi cv. ICC 15061). The QTL-seq-derived major SW QTL (CaqSW1.1) was further validated by single-nucleotide polymorphism (SNP) and simple sequence repeat (SSR) marker-based traditional QTL mapping (47.6% R² at higher LOD >19). This reflects the reliability and efficacy of QTL-seq as a strategy for rapid genome-wide scanning and fine mapping of major trait regulatory QTLs in chickpea. The use of QTL-seq and classical QTL mapping in combination narrowed down the 1.37 Mb (comprising 177 genes) major SW QTL (CaqSW1.1) region into a 35 kb genomic interval on desi chickpea chromosome 1 containing six genes. One coding SNP (G/A)-carrying constitutive photomorphogenic9 (COP9) signalosome complex subunit 8 (CSN8) gene of these exhibited seed-specific expression, including pronounced differential up-/down-regulation in low and high seed weight mapping parents and homozygous individuals during seed development. The coding SNP mined in this potential seed weight-governing candidate CSN8 gene was found to be present exclusively in all cultivated species/genotypes, but not in any wild species/genotypes of primary, secondary and tertiary gene pools. This indicates the effect of strong artificial and/or natural selection pressure on target SW locus during chickpea domestication. The proposed QTL-seq-driven integrated genome-wide strategy has potential to delineate major candidate gene(s) harbouring a robust trait regulatory QTL rapidly with optimal use of resources. This will further assist us to extrapolate the molecular mechanism underlying complex quantitative traits at a genome-wide scale leading to fast-paced marker-assisted genetic improvement in diverse crop plants, including chickpea.     

High-Throughput SNP Discovery and Genotyping for Constructing a Saturated Linkage Map of Chickpea (Cicer arietinum L.)

The present study reports the large-scale discovery of genome-wide single-nucleotide polymorphisms (SNPs) in chickpea, identified mainly through the next generation sequencing of two genotypes, i.e. Cicer arietinum ICC4958 and its wild progenitor C. reticulatum PI489777, parents of an inter-specific reference mapping population of chickpea. Development and validation of a high-throughput SNP genotyping assay based on Illumina''s GoldenGate Genotyping Technology and its application in building a high-resolution genetic linkage map of chickpea is described for the first time. In this study, 1022 SNPs were identified, of which 768 high-confidence SNPs were selected for designing the custom Oligo Pool All (CpOPA-I) for genotyping. Of these, 697 SNPs could be successfully used for genotyping, demonstrating a high success rate of 90.75%. Genotyping data of the 697 SNPs were compiled along with those of 368 co-dominant markers mapped in an earlier study, and a saturated genetic linkage map of chickpea was constructed. One thousand and sixty-three markers were mapped onto eight linkage groups spanning 1808.7 cM (centiMorgans) with an average inter-marker distance of 1.70 cM, thereby representing one of the most advanced maps of chickpea. The map was used for the synteny analysis of chickpea, which revealed a higher degree of synteny with the phylogenetically close Medicago than with soybean. The first set of validated SNPs and map resources developed in this study will not only facilitate QTL mapping, genome-wide association analysis and comparative mapping in legumes but also help anchor scaffolds arising out of the whole-genome sequencing of chickpea.     

Inheritance of growth habit detected by genetic linkage analysis using microsatellites in the common bean (Phaseolus vulgaris L.)

The genetic linkage map for the common bean (Phaseolus vulgaris L.) is a valuable tool for breeding programs. Breeders provide new cultivars that meet the requirements of farmers and consumers, such as seed color, seed size, maturity, and growth habit. A genetic study was conducted to examine the genetics behind certain qualitative traits. Growth habit is usually described as a recessive trait inherited by a single gene, and there is no consensus about the position of the locus. The aim of this study was to develop a new genetic linkage map using genic and genomic microsatellite markers and three morphological traits: growth habit, flower color, and pod tip shape. A mapping population consisting of 380 recombinant F10 lines was generated from IAC-UNA × CAL143. A total of 871 microsatellites were screened for polymorphisms among the parents, and a linkage map was obtained with 198 mapped microsatellites. The total map length was 1865.9 cM, and the average distance between markers was 9.4 cM. Flower color and pod tip shape were mapped and segregated at Mendelian ratios, as expected. The segregation ratio and linkage data analyses indicated that the determinacy growth habit was inherited as two independent and dominant genes, and a genetic model is proposed for this trait.     

Genetic variation of a global germplasm collection of chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.) including Italian accessions at risk of genetic erosion

Chickpea (Cicer arietinum L.) is one of the most important legumes worldwide. We addressed this study to the genetic characterization of a germplasm collection from main chickpea growing countries. Several Italian traditional landraces at risk of genetic erosion were included in the analysis. Twenty-two simple sequence repeat (SSR) markers, widely used to explore genetic variation in plants, were selected and yielded 218 different alleles. Structure analysis and hierarchical clustering indicated that a model with three distinct subpopulations best fits the data. The composition of two subpopulations, named K1 and K2, broadly reflects the commercial classification of chickpea in the two types desi and kabuli, respectively. The third subpopulation (K3) is composed by both desi and kabuli genotypes. Italian accessions group both in K2 and K3. Interestingly, this study highlights genetic distance between desi genotypes cultivated in Asia and Ethiopia, which respectively represent the chickpea primary and the secondary centres of diversity. Moreover, European desi are closer to the Ethiopian gene pool. Overall, this study will be of importance for chickpea conservation genetics and breeding, which is limited by the poor characterization of germplasm collection.     

Use of VeraCode 384-plex assays for watermelon diversity analysis and integrated genetic map of watermelon with single nucleotide polymorphisms and simple sequence repeats

Watermelon (Citrullus lanatus var. lanatus) is one of the most important vegetable crops in the world. Molecular markers have become the tools of choice for resolving watermelon taxonomic relationships and evolution. Increased numbers of single nucleotide polymorphism (SNP) markers together with simple sequence repeat (SSR) markers would be useful for phylogenetic analyses of germplasm accessions and for linkage mapping for marker-assisted breeding with quantitative trait loci and single genes. We aimed to construct a genetic map based on SNPs (generated by Illumina Veracode multiplex assays for genotyping) and SSR markers and evaluate relationships inferred from SNP genotypes between 130 watermelon accessions collected throughout the world. We incorporated 282 markers (232 SNPs and 50 SSRs) into the linkage map. The genetic map consisted of 11 linkage groups spanning 924.72 cM with an average distance of 3.28 cM between markers. Because all of the SNP-containing sequences were assembled with the whole-genome sequence draft for watermelon, chromosome numbers could be readily assigned for all the linkage groups. We found that 134 SNPs were polymorphic in 130 watermelon accessions chosen for diversity studies. The current 384-plex SNP set is a powerful tool for characterizing genetic relatedness and for developing medium-resolution genetic maps.     

Construction of an SSR and RAD Marker-Based Genetic Linkage Map for Carnation (<Emphasis Type="Italic">Dianthus caryophyllus</Emphasis> L.)

A high-density genetic map, an essential tool for comparative genomic studies and quantitative trait locus fine mapping, can also facilitate genome sequence assembly. The sequence-based marker technology known as restriction site-associated DNA (RAD) enables synchronous, single nucleotide polymorphism marker discovery, and genotyping using massively parallel sequencing. We constructed a high-density linkage map for carnation (Dianthus caryophyllus L.) based on simple sequence repeat (SSR) markers in combination with RAD markers developed by double-digest RAD sequencing (ddRAD-seq). A total of 2404 (285 SSR and 2119 RAD) markers could be assigned to 15 linkage groups spanning 971.5 cM, with an average marker interval of 0.4 cM. The total length of scaffolds with identified map positions was 95.6 Mb, which is equivalent to 15.4 % of the estimated genome size. The generated map is the first SSR and RAD marker-based high-density linkage map reported for carnation. The ddRAD-seq pipeline developed in this study should also help accelerate genetic and genomics analyses and molecular breeding of carnation and other non-model crops.     

SRAP-based mapping and QTL detection for inflorescence-related traits in chrysanthemum (Dendranthema morifolium)

Chrysanthemum (Dendranthema morifolium) is an economically important ornamental species and comprises a large proportion of the flower industry in south-east Asian and European countries. In this study, a segregating population of 142 F₁ progeny of the cross between the two chrysanthemum cultivars ??Yuhualuoying?? and ??Aoyunhanxiao?? was used to construct two separate genetic linkage maps via a double pseudo-testcross mapping strategy. Genotyping was performed using 500 SRAP primer combinations, of which about 50% were informative. This allowed the definition of 896 SRAP loci, of which about 23% showed some segregation distortion. The ??Yuhualuoying?? map consisted of 333 testcross markers arranged into 57 linkage groups (LGs). It covered >1,900 cM with a mean inter-marker distance of 6.9 cM. The map constructed from ??Aoyunhanxiao?? comprised 342 test cross markers arranged into 55 LGs. It spanned nearly 1,900 cM, with a mean inter-marker distance of 6.6 cM. The markers were distributed rather uniformly along both maps. A quantitative trait loci analysis was conducted to investigate the pattern of inheritance of three inflorescence traits. This led to the detection of 12 putative loci at a LOD score >2.5, of which four each specified flower diameter, ray floret layer number, and ray floret length. This study provides molecular mapping information on marker-assisted selection programs for the improvement of multiple traits of interest.     

Colchicine induced polyploidy in chickpeas (Cicer arietinum L.)

Summary The most successful induction of tetraploidy was obtained with 2 hour treatment by 0.25% aqueous colchicine solution of 18-hour watersoaked desi chickpeas material. However, kabuli types needed only 1 hour treatment under similar conditions. Gigantism accompanied induction of polyploidy in desi as well as kabuli types but yield and fertility were greatly reduced. The meiotic abnormalities accompanying polyploidy were multivalent association of chromosomes followed by unequal disjunction, chromosome bridges, laggards etc. The percentage of stainable pollen, however, was at par between diploids and tetraploids. Gene control of percentage seed setting was observed in both levels of ploidy. A striking feature of the studies was the high seed setting percentage in 4n F ₁ material resulting from diverse crosses, viz., desi×kabuli. A probable reduction in multivalent association coupled with yield increases in segregants from the later generation of tetraploids indicates the possibility of selection for higher yield and fertility from polyploids, particularly from some hybrid material.     

Narrow Genetic Variability in Cicer arietinum L. as Revealed by RFLP Analysis

A subgenomic library constructed from small Pstl restriction fragments (0.4 to 2.0 kb) yielded 83.18% low-copy clones. Using 17 random genomic and 5 heterologous probes in 65 probe-enzyme combinations, Restriction Fragment Length Polymorphism (RFLP) for nuclear DNA was studied in five desi and five kabuli type chickpea cultivars. Only two clones revealed polymorphism in the cultivars tested. No polymorphism in chickpea varieties was detected with four Random Amplified Polymorphic DNA (RAPD) markers studied. However, some degree of polymorphism between C. arietinum and its wild relative C. reticulatum was detected. The RFLP analysis of chloroplast and mitochondrial genomes showed no polymorphism.     

A SNP Based High-Density Linkage Map of Apis cerana Reveals a High Recombination Rate Similar to Apis mellifera

Background

The Eastern honey bee, Apis cerana Fabricius, is distributed in southern and eastern Asia, from India and China to Korea and Japan and southeast to the Moluccas. This species is also widely kept for honey production besides Apis mellifera. Apis cerana is also a model organism for studying social behavior, caste determination, mating biology, sexual selection, and host-parasite interactions. Few resources are available for molecular research in this species, and a linkage map was never constructed. A linkage map is a prerequisite for quantitative trait loci mapping and for analyzing genome structure. We used the Chinese honey bee, Apis cerana cerana to construct the first linkage map in the Eastern honey bee.

Results

F2 workers (N = 103) were genotyped for 126,990 single nucleotide polymorphisms (SNPs). After filtering low quality and those not passing the Mendel test, we obtained 3,000 SNPs, 1,535 of these were informative and used to construct a linkage map. The preliminary map contains 19 linkage groups, we then mapped the 19 linkage groups to 16 chromosomes by comparing the markers to the genome of A. mellfiera. The final map contains 16 linkage groups with a total of 1,535 markers. The total genetic distance is 3,942.7 centimorgans (cM) with the largest linkage group (180 loci) measuring 574.5 cM. Average marker interval for all markers across the 16 linkage groups is 2.6 cM.

Conclusion

We constructed a high density linkage map for A. c. cerana with 1,535 markers. Because the map is based on SNP markers, it will enable easier and faster genotyping assays than randomly amplified polymorphic DNA or microsatellite based maps used in A. mellifera.     

Characterization of the quantitative trait locus OilA1 for oil content in Brassica napus

Increasing seed oil content has become one of the most important breeding criteria in rapeseed (Brassica napus). However, oil content is a complex quantitative trait. QTL mapping in a double haploid population (SG population) emerging from a cross between a German (Sollux) and Chinese (Gaoyou) cultivars revealed one QTL for oil content on linkage group A1 (OilA1), which was mapped to a 17 cM genetic interval. To further validate and characterize the OilA1, we constructed a high-resolution map using B. rapa sequence resources and developed a set of near-isogenic lines (NILs) by employing a DH line SG-DH267 as donor and Chinese parent Gaoyou as recurrent background. The results showed highly conserved synteny order between B. rapa and B. napus within the linkage group A1 and revealed a possible centromere region between two markers ZAASA1-38 and NTP3 (2.5 cM). OilA1 was firstly validated by 250 BC₅F₂ plants and was confirmed in a 10.6 cM interval between the markers ZAASA1-47 and ZAASA1-77. Further substitution mapping was conducted by using two generations of QTL-NILs, 283 lines from eight BC₅F_3:4 families and 428 plants from six BC₅F₄ sub-NILs and thus narrowed the OilA1 interval to 6.9 cM and 4.3 cM (1.4 Mb), respectively. Field investigations with two replications using homozygous BC₅F_3:4 sister sub-NILs indicated that NILs, which carry a Sollux chromosome segment across the target region showed significant higher oil content (1.26 %, p < 0.001) than their sister NILs containing Gaoyou chromosome. The OilA1 locus is of particular interest for breeding purpose in China because 80 % of Chinese cultivars do not carry this desirable allele.