The first draft of the pigeonpea genome sequence

Nagendra K. Singh 0 1 2 3 Deepak K. Gupta 0 1 2 3 Pawan K. Jayaswal 0 1 2 3 Ajay K. Mahato 0 1 2 3 Sutapa Dutta 0 1 2 3 Sangeeta Singh 0 1 2 3 Shefali Bhutani 0 1 2 3 Vivek Dogra 0 1 2 3 Bikram P. Singh 0 1 2 3 Giriraj Kumawat 0 1 2 3 Jitendra K. Pal 0 1 2 3 Awadhesh Pandit 0 1 2 3 Archana Singh 0 1 2 3 Hukum Rawal 0 1 2 3 Akhilesh Kumar 0 1 2 3 G. Rama Prashat 0 1 2 3 Ambika Khare 0 1 2 3 Rekha Yadav 0 1 2 3 Ranjit S. Raje 0 1 2 3 Mahendra N. Singh 0 1 2 3 Subhojit Datta 0 1 2 3 Bashasab Fakrudin 0 1 2 3 Keshav B. Wanjari 0 1 2 3 Rekha Kansal 0 1 2 3 Prasanta K. Dash 0 1 2 3 Pradeep K. Jain 0 1 2 3 Ramcharan Bhattacharya 0 1 2 3 Kishor Gaikwad 0 1 2 3 Trilochan Mohapatra 0 1 2 3 R. Srinivasan 0 1 2 3 Tilak R. Sharma 0 1 2 3 0 M. N. Singh Institute of Agricultural Sciences, Banaras Hindu University , Varanasi, UP 221005, India 1 K. B. Wanjari Panjabrao Deshmukh Krishi Vidyapeeth, Krishinagar, Akola, Maharasthra 444 104, India 2 B. Fakrudin University of Agricultural Sciences , Dharwad, Karnataka 580005, India 3 S. Datta Indian Institute of Pulses Research , Kanpur, UP 208024, India Pigeonpea (Cajanus cajan) is an important grain legume of the Indian subcontinent, South-East Asia and East Africa. More than eighty five percent of the world pigeonpea is produced and consumed in India where it is a - key crop for food and nutritional security of the people. Here we present the first draft of the genome sequence of a popular pigeonpea variety Asha. The genome was assembled using long sequence reads of 454 GS-FLX sequencing chemistry with mean read lengths of >550 bp and >10-fold genome coverage, resulting in 510,809,477 bp of high quality sequence. Total 47,004 protein coding genes and 12,511 transposable elements related genes were predicted. We identified 1,213 disease resistance/defense response genes and 152 abiotic stress tolerance genes in the pigeonpea genome that make it a hardy crop. In comparison to soybean, pigeonpea has relatively fewer number of genes for lipid biosynthesis and larger number of genes for cellulose synthesis. The sequence contigs were arranged in to 59,681 scaffolds, which were anchored to eleven chromosomes of pigeonpea with 347 genic-SNP markers of an intra-species reference genetic map. Eleven pigeonpea chromosomes showed low but significant synteny with the twenty chromosomes of soybean. The genome sequence was used to identify large number of hypervariable Arhar simple sequence repeat (HASSR) markers, 437 of which were experimentally validated for PCR amplification and high rate of polymorphism among pigeonpea varieties. These markers will be useful for fingerprinting and diversity analysis of pigeonpea germplasm and molecular breeding applications. This is the first plant genome sequence completed entirely through a network of Indian institutions led by the Indian Council of Agricultural Research and provides a valuable resource for the pigeonpea variety improvement. Abbreviations HASSR hypervariable Arhar simple sequence repeats SNP single nucleotide polymorphism AKI Agricultural knowledge initiative Pigeonpea or Red Gram (Cajanus cajan (L.) Millspaugh) is an important food legume for the tropical and subtropical regions of Indian subcontinent, South-East Asia and East Africa. It is a shrub with self-compatible cleistogamous flowers, but is often cross-pollinated by bees with 1015% out crossing. The estimated size of pigeonpea genome packed in 11 chromosomes is 858 Mbp (Greilhuber and Obermayer 1998). It plays important role in food and nutritional security because it is a rich source of proteins, minerals and vitamins. Pigeonpea seeds are consumed mainly as split pea soups such as Dal and Sambar but a significant proportion is also consumed as green pea vegetable and whole grain preparations. Its leaves, seed husks and pod husks are used as animal feed (Fig. 1). Symbiotic bacteria (Bradyrhizobium) colonizing root nodules of pigeonpea fix atmospheric nitrogen up to 40 kg/ha in a cropping season and its deep root system improves soil structure and organic matter. Pigeonpea is unique among the legume crops as it is a woody shrub, therefore its stem and branches are used for firewood, fencing, thatch and making baskets by the rural population. Archeological evidence indicates that pigeonpea was domesticated in the eastern part of the Indian subcontinent along with rice and other important grain legumes, namely Urd or black gram (Vigna mungo), Mung or green gram (Vigna radiata) and Kulthi or horse gram (Macrotyloma uniflorum) during prehistoric period (Fuller 2006). The world acreage of pigeonpea is 4.90 mha with annual production of about 4.22 mmt worth about 1.5 billion US dollars. India is the largest producer and consumer of pigeonpea (local names Arhar, Tur) with annual production of 3.07 mmt, followed by Myanmar (0.72 mmt) and Malawi (0.15 mmt) (FAOSTAT 2008). Fig. 1 The whole plant and different parts of the pigeonpea cultivar Asha (ICPL 87119). a whole plant at fruiting stage; b a defoliated branch with pods; c a branch with heavy flowering; d mature seeds; e dehusked split seeds or Dal; f 22 chromosomes in a root tip cell Knowledge of the genetic basis of yield, quality and stress tolerance is important for genetic improvement of pigeonpea. Until a couple of years ago pigeonpea was considered an orphan legume crop but now substantial amount of genomic resources have been generated, largely owing to the efforts of Indo-US Agricultural Knowledge Initiative (AKI), NSF and GCP funded projects, (Varshney et al. 2009, 2010a; Dutta et al. 2011; Bohra et al. 2011). Pigeonpea cultivars have a narrow genetic base due to limited breeding efforts and poor utilization of wild pigeonpea species. Availability of genome sequence will accelerate the utilization of pigeonpea germplasm resources in breeding (Yang et al. 2006; Saxena 2008; Varshney et al. 2010b). Development of molecular markers tightly linked to the important agronomic traits is a prerequisite for undertaking molecular breeding in plants. But molecular basis of most agronomic traits in pigeonpea remains unexplored due to low level of DNA polymorphism in the primary gene pool and limited number of validated molecular markers (Ratnaparkhe et al. 1995; Yang et al. 2006; Odeny et al. 2009; Dutta et al. 2011; Bohra et al. 2011). The aim of present study was: (a) to decode the pigeonpea genome by using next generation sequencing technologies and analyse its genes and repeat DNA contents; (b) generation of chromosome specific sequence by anchoring the sequence scaffolds to a high density reference molecular linkage map and its comparison with soybean genome; and (c) development of SSR markers for gene discovery and molecular breeding applications. Pigeonpea variety Asha selected for this purpose is a popular variety with one of the highest breeder seed indents in India and is resistant to common diseases of pigeonpea, namely Fusarium wilt and sterility mosaic disease. Materials and meth (...truncated)