Generation of Expressed Sequence Tags and Marker Development for Sterility Mosaic Disease Resistance in Pigeonpea

Sterility mosaic disease (SMD), an important biotic constraint on pigeonpea (Cajanus cajan (L) Millsp.) in the Indian subcontinent, is caused by Pigeonpea sterility mosaic virus and transmitted by the eriophyid mite. Investigations were carried out to develop F2:3 mapping population involving resistant (ICP 7035) and susceptible (TTB 7) parents, development of EST SSR markers, construction of linkage map and identification of QTLs for SMD resistance. F1s of the susceptible × resistant cross were susceptible indicating susceptibility to be dominant over resistance. The pattern of frequency distribution of SMD incidence in the F2:3 was found to be continuous depicting quantitative nature of resistance. Totally 3,788 high quality ESTs were generated from SMD challenged genotypes of ICP 7035 and TTB 7. Sequence clustering and assembly process of all the assembled 3,788 ESTs resulted in 1,308 unigenes. Out of 3320 SSR markers (3236 genomic+84 genic) screened for SMD in TTB 7 and ICP 7035, 2107 (63.5%) could amplify and 84 (83 genomic + 01 genic) SSR markers were found to be polymorphic (2.5%). A total of 82 markers were mapped on 11 linkage groups (LGs) of pigeonpea spanning 539.5 cM and two markers remained ungrouped. Number of markers mapped per linkage group ranged from three (LG 11) to twelve (LG 7). The present study yielded two QTLs for Bengaluru isolate of SMD positioned on LG 3 and LG 7 accounting 10.39 per cent and 15.74 of the phenotypic variation respectively. For Patancheru isolate also, two QTLs were identified and the first QTL explained 12.3 per cent phenotypic variation and the second QTL explained 24.69 per cent of phenotypic variation

Inheritance of sterility mosaic disease resistance to Bangalore and Patancheru isolates in pigeonpea (Cajanus cajan (L.) Millsp.)

Sterility mosaic disease (SMD), is an important biotic constraint in pigeonpea (Cajanus cajan (L.) Millsp.) in Indian subcontinent. It is caused by a virus and transmitted by eriophyid mites, Aceria cajani Channabasavanna. A comprehensive study of variability in the sterility mosaic pathogen revealed the occurrence of five different isolates in India. Amongst them, three distinct isolates have been characterised, viz., Bangalore, Patancheru and Coimbatore. Studies were conducted at Bangalore and Patancheru to determine the inheritance of resistance to Bangalore and Patancheru isolates of the SMD involving a resistant (ICP 7035) and susceptible (TTB 7) genotypes. Observations in parents, F indicated dominance of susceptibility over resistance. The disease reaction of the individual F 2 plant derived F 3 1 families for Patancheru isolate was controlled by two genes with dominance epistasis and for Bangalore isolate, absence of resistant plants indicate action of two or more genes in controlling resistance to SMD

The first set of EST resource for gene discoveryand marker development in pigeonpea(Cajanus cajan L.)

Pigeonpea (Cajanus cajan (L.) Millsp) is one of the major grain legume crops of the tropics and subtropics, but biotic stresses [Fusarium wilt (FW), sterility mosaic disease (SMD), etc.] are serious challenges for sustainable crop production. Modern genomic tools such as molecular markers and candidate genes associated with resistance to these stresses offer the possibility of facilitating pigeonpea breeding for improving biotic stress resistance. Availability of limited genomic resources, however, is a serious bottleneck to undertake molecular breeding in pigeonpea to develop superior genotypes with enhanced resistance to above mentioned biotic stresses. With an objective of enhancing genomic resources in pigeonpea, this study reports generation and analysis of comprehensive resource of FW- and SMD- responsive expressed sequence tags (ESTs). Results: A total of 16 cDNA libraries were constructed from four pigeonpea genotypes that are resistant and susceptible to FW ('ICPL 20102' and 'ICP 2376') and SMD ('ICP 7035' and 'TTB 7') and a total of 9,888 (9,468 high quality) ESTs were generated and deposited in dbEST of GenBank under accession numbers GR463974 to GR473857 and GR958228 to GR958231. Clustering and assembly analyses of these ESTs resulted into 4,557 unique sequences (unigenes) including 697 contigs and 3,860 singletons. BLASTN analysis of 4,557 unigenes showed a significant identity with ESTs of different legumes (23.2-60.3%), rice (28.3%), Arabidopsis (33.7%) and poplar (35.4%). As expected, pigeonpea ESTs are more closely related to soybean (60.3%) and cowpea ESTs (43.6%) than other plant ESTs. Similarly, BLASTX similarity results showed that only 1,603 (35.1%) out of 4,557 total unigenes correspond to known proteins in the UniProt database (= 1E-08). Functional categorization of the annotated unigenes sequences showed that 153 (3.3%) genes were involved in cellular component category, 132 (2.8%) in biological process, and 132 (2.8%) in molecular function. Further, nineteen genes were identified differentially expressed between FW- responsive genotypes and 20 between SMD- responsive genotypes. Generated ESTs were compiled together with 908 ESTs available in public domain, at the time of analysis, and a set of 5,085 unigenes were defined that were used for identification of molecular markers in pigeonpea. For instance, 3,583 simple sequence repeat (SSR) motifs were identified in 1,365 unigenes and 383 primer pairs were designed. Assessment of a set of 84 primer pairs on 40 elite pigeonpea lines showed polymorphism with 15 (28.8%) markers with an average of four alleles per marker and an average polymorphic information content (PIC) value of 0.40. Similarly, in silico mining of 133 contigs with ⩾ 5 sequences detected 102 single nucleotide polymorphisms (SNPs) in 37 contigs. As an example, a set of 10 contigs were used for confirming in silico predicted SNPs in a set of four genotypes using wet lab experiments. While occurrence of SNPs were confirmed for all the 6 contigs for which scorable and sequenceable amplicons were generated. PCR amplicons were not obtained in case of 4 contigs. Recognition sites for restriction enzymes were identified for 102 SNPs in 37 contigs that indicates possibility of assaying SNPs in 37 genes using cleaved amplified polymorphic sequences (CAPS) assa

An intra-specific consensus genetic map of pigeonpea [Cajanus cajan (L.) Millspaugh] derived from six mapping populations

Pigeonpea (Cajanus cajan L.) is an important food legume crop of rainfed agriculture. Owing to exposure of the crop to a number of biotic and abiotic stresses, the crop productivity has remained stagnant for almost last five decades at ca. 750 kg/ha. The availability of a cytoplasmic male sterility (CMS) system has facilitated the development and release of hybrids which are expected to enhance the productivity of pigeonpea. Recent advances in genomics and molecular breeding such as marker-assisted selection (MAS) offer the possibility to accelerate hybrid breeding. Molecular markers and genetic maps are pre-requisites for deploying MAS in breeding. However, in the case of pigeonpea, only one inter- and two intra-specific genetic maps are available so far. Here, four new intra-specific genetic maps comprising 59–140 simple sequence repeat (SSR) loci with map lengths ranging from 586.9 to 881.6 cM have been constructed. Using these four genetic maps together with two recently published intra-specific genetic maps, a consensus map was constructed, comprising of 339 SSR loci spanning a distance of 1,059 cM. Furthermore, quantitative trait loci (QTL) analysis for fertility restoration (Rf) conducted in three mapping populations identified four major QTLs explaining phenotypic variances up to 24 %. To the best of our knowledge, this is the first report on construction of a consensus genetic map in pigeonpea and on the identification of QTLs for fertility restoration. The developed consensus genetic map should serve as a reference for developing new genetic maps as well as correlating with the physical map in pigeonpea to be developed in near future. The availability of more informative markers in the bins harbouring QTLs for sterility mosaic disease (SMD) and Rf will facilitate the selection of the most suitable markers for genetic analysis and molecular breeding applications in pigeonpea

A Consensus Map in Cultivated Hexaploid Oat Reveals Conserved Grass Synteny with Substantial Subgenome Rearrangement

Citation: Chaffin, A. S., Huang, Y. F., Smith, S., Bekele, W. A., Babiker, E., Gnanesh, B. N., . . . Tinker, N. A. (2016). A Consensus Map in Cultivated Hexaploid Oat Reveals Conserved Grass Synteny with Substantial Subgenome Rearrangement. Plant Genome, 9(2), 21. doi:10.3835/plantgenome2015.10.0102Hexaploid oat (Avena sativa L., 2n = 6x = 42) is a member of the Poaceae family and has a large genome (similar to 12.5 Gb) containing 21 chromosome pairs from three ancestral genomes. Physical rearrangements among parental genomes have hindered the development of linkage maps in this species. The objective of this work was to develop a single high-density consensus linkage map that is representative of the majority of commonly grown oat varieties. Data from a cDNA-derived single-nucleotide polymorphism (SNP) array and genotyping-by-sequencing (GBS) were collected from the progeny of 12 biparental recombinant inbred line populations derived from 19 parents representing oat germplasm cultivated primarily in North America. Linkage groups from all mapping populations were compared to identify 21 clusters of conserved collinearity. Linkage groups within each cluster were then merged into 21 consensus chromosomes, generating a framework consensus map of 7202 markers spanning 2843 cM. An additional 9678 markers were placed on this map with a lower degree of certainty. Assignment to physical chromosomes with high confidence was made for nine chromosomes. Comparison of homeologous regions among oat chromosomes and matches to orthologous regions of rice (Oryza sativa L.) reveal that the hexaploid oat genome has been highly rearranged relative to its ancestral diploid genomes as a result of frequent translocations among chromosomes. Heterogeneous chromosome rearrangements among populations were also evident, probably accounting for the failure of some linkage groups to match the consensus. This work contributes to a further understanding of the organization and evolution of hexaploid grass genomes

Advances in genetics and molecular breeding of three legume crops of semi-arid tropics using next-generation sequencing and high-throughput genotyping technologies

Molecular markers are the most powerful genomic tools to increase the efficiency and precision of breeding practices for crop improvement. Progress in the development of genomic resources in the leading legume crops of the semi-arid tropics (SAT), namely, chickpea (Cicer arietinum), pigeonpea (Cajanus cajan) and groundnut (Arachis hypogaea), as compared to other crop species like cereals, has been very slow. With the advances in next-generation sequencing (NGS) and high-throughput (HTP) genotyping methods, there is a shift in development of genomic resources including molecular markers in these crops. For instance, 2,000 to 3,000 novel simple sequence repeats (SSR) markers have been developed each for chickpea, pigeonpea and groundnut. Based on Sanger, 454/FLX and Illumina transcript reads, transcriptome assemblies have been developed for chickpea (44,845 transcript assembly contigs, or TACs) and pigeonpea (21,434 TACs). Illumina sequencing of some parental genotypes of mapping populations has resulted in the development of 120 million reads for chickpea and 128.9 million reads for pigeonpea. Alignment of these Illumina reads with respective transcriptome assemblies have provided >10,000 SNPs each in chickpea and pigeonpea. A variety of SNP genotyping platforms including GoldenGate, VeraCode and Competitive Allele Specific PCR (KASPar) assays have been developed in chickpea and pigeonpea. By using above resources, the first-generation or comprehensive genetic maps have been developed in the three legume speciesmentioned above. Analysis of phenotyping data together with genotyping data has provided candidate markers for drought-tolerance-related root traits in chickpea, resistance to foliar diseases in groundnut and sterility mosaic disease (SMD) and fertility restoration in pigeonpea. Together with these traitassociated markers along with those already available, molecular breeding programmes have been initiated for enhancing drought tolerance, resistance to fusarium wilt and ascochyta blight in chickpea and resistance to foliar diseases in groundnut. These trait-associated robust markers along with other genomic resources including genetic maps and genomic resources will certainly accelerate crop improvement programmes in the SAT legum

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Not AvailablePigeonpea is one of the important legume crops of India which is affected by Fusarium wilt (Fusarium udum) disease causing severe yield loss. Four different races of Fusarium wilt have reported been with pathogenic race present in Bangalore being most virulent. Hence in the present study nature of inheritance of wilt disease was studied in segregating generations (F2 and F3) of crosses namely BRG-1 × ICP-8863 and TTB-7 × ICP-8863. Digenic ratio of 9 (susceptible): 7 (resistant) and 13 (susceptible): 3 (resistant) was obtained in F2 generation of two crosses BRG-1 × ICP-8863 and TTB-7 × ICP-8863, respectively. Frequency distribution of F3 generation showed normal curve, skewed towards susceptibility. This indicates that susceptibility was dominant over resistance and is governed by two or more genes. Probable loci responsible for disease reaction have been designated as FuB1, FuB2 and FuB3. Susceptible parents (TTB 7 and BRG 1) shared one common dominant gene whereas ICP 8863 had recessive resistant gene. Characterisation of these genes will help in marker assisted breeding programmeNot Availabl

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Not AvailableAn experiment was conducted to identify markers linked to Fusarium wilt disease resistance, Parents namely TTB 7 and ICP 8863 were screened using 151 SSRs markers and 16 AFLP primer combinations. Parental screening revealed five SSR primers and 12 AFLP primer combinations polymorphic between parents. Bulk segregant analysis identified five AFLP primer combinations generating seven markers polymorphic between resistant and susceptible bulks while, none of the SSR markers were polymorphic. This indicates that, these markers are putatively linked to wilt disease. Screening of F2 segregating population of cross TTB 7 x ICP 8863 with these putatively linked markers revealed four markers (E-AAT/M-CTG850, ETCG/ M-CTT650, E-TCG/M-CTA730 and E-TCG/M-CTT230) which segregated in 3:1 mendelian pattern. Simple linear regression performed on these four markers had identified two markers namely E-TCG/M-CTT650 and E-TCG/M-CTA730 linked to diseaseNot Availabl