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Production and Cytological Characterization of Oryza Sativa and Oryza Punctata Derived Synthetic Amphiploids

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Production and Cytological Characterization of Oryza Sativa and Oryza Punctata Derived Synthetic Amphiploids Genome Production and cytological characterization of Oryza sativa and Oryza punctata derived synthetic amphiploids Journal: Genome Manuscript ID gen-2019-0062.R1 Manuscript Type: Article Date Submitted by the 28-Jun-2019 Author: Complete List of Authors: Kumar, Kishor; Punjab Agricultural University, School of Agricultural Biotechnology Neelam, Kumari; Punjab Agricultural University, School of Agricultural Biotechnology Singh, Gurpreet;Draft Punjab Agricultural University, School of Agricultural Biotechnology Mathan, Jyotirmaya; NIPGR Ranjan, Aashish; NIPGR Brar, Darshan; Punjab Agricultural University, School of Agricultural Biotechnology Singh, Kuldeep; Punjab Agricultural University, School of Agricultural Biotechnology; National Bureau of Plant Genetic Resources Oryza punctata, Synthetic Amphiploids, Aneuploids, Cytology, Flow Keyword: Cytometery Is the invited manuscript for consideration in a Special Not applicable (regular submission) Issue? : https://mc06.manuscriptcentral.com/genome-pubs Page 1 of 28 Genome 1 Production and cytological characterization of Oryza sativa and Oryza punctata derived synthetic 2 amphiploids 3 Kishor Kumar1,3, Kumari Neelam1,#, Gurpreet Singh1 , Jyotirmaya Mathan2, Aashish Ranjan2 Darshan 4 Singh Brar1 and Kuldeep Singh1,4 5 1 School of Agricultural Biotechnology, Punjab Agricultural University, Ludhiana, Punjab, 141004, 6 India. 7 2 National Institute of Plant Genome Research, New Delhi, 110067, India 8 3 Faculty Centre on Integrated Rural Development and Management, Ramakrishna Mission 9 Vivekanada Educational and Research Institute, Narendrapur, Kolkata, 700103, India 10 4 ICAR- National Bureau of Plant Genetic Resources, PUSA, New Delhi, 110012, India 11 12 # Author for correspondence 13 Kumari Neelam Draft 14 School of Agricultural Biotechnology, Punjab Agricultural University, Ludhiana, Punjab, 141004, 15 India 16 Email: [email protected] 17 Mobile No.: +917986044964 18 19 20 21 22 23 24 25 26 27 28 1 https://mc06.manuscriptcentral.com/genome-pubs Genome Page 2 of 28 29 Abstract 30 Oryza punctata Kotschy ex Steud. (BB, 2n=24) is a wild species of rice having many useful 31 agronomic traits. An interspecific hybrid (AB, 2n=24) was produced by crossing O. punctata and O. 32 sativa cv. Punjab Rice 122 (PR122, AA, 2n= 24) to broaden the narrow genetic base of cultivated 33 rice. Cytological analysis of the pollen mother cells (PMCs) of interspecific hybrids confirmed 24 34 chromosomes. The F1 hybrids showed the presence of 19-20 univalents and 1-3 bivalents. The 35 interspecific hybrid was treated with colchicine to produce synthetic amphiploid (AABB, 2n=48). 36 Pollen fertility of synthetic amphiploid was found more than 50% and partial seed set was observed. 37 Chromosome numbers in the PMCs of synthetic amphiploid were 24II showing normal pairing. Flow 38 cytometric analysis also confirmed doubled genomic content in the synthetic amphiploid than diploid. 39 Leaf morphological and anatomical studies of synthetic amphiploid showed higher chlorophyll 40 content and enlarge bundle sheath cells as compared to both of its parents. The synthetic amphiploid 41 was backcrossed with PR122 to develop aDraft series of addition and substitution lines for the transfer of 42 useful genes from O. punctata with least linkage drag. 43 Keywords 44 Oryza punctata, Synthetic Amphiploids, Aneuploids, Cytology, Flow Cytometery, Chromosome 45 Doubling 46 47 48 49 50 51 52 53 54 55 56 2 https://mc06.manuscriptcentral.com/genome-pubs Page 3 of 28 Genome 57 Introduction 58 Rice (Oryza sativa L.) is one of the most important staple food crop and source of calories for 59 mankind worldwide. The genus Oryza of the Gramineae family has 24 species, of which 22 are wild 60 species and two (O. sativa L. and O. glaberrima Steud.) are cultivated (Khush 1997, Vaughan 1989). 61 Wild species are inferior in growth and weedy in nature but are the reservoir for many useful genes 62 that can be used in the modern breeding programs to enhance yield potential and resistance (Brar and 63 Khush 1997; Jena 2010). Domestication leads to loss of many agronomically important genes and 64 hence, limited genetic variability available in the cultivated gene pool of rice (Tanksley and McCouch 65 1997). Identification and exploitation of the genes from wild species of rice are necessary to 66 overcome genetic bottleneck and broaden the narrow genetic base, as they have accumulated 67 abundant genetic diversity. Transfer of genes from secondary and tertiary gene pools to primary gene 68 pools through conventional breeding method is a herculean task, because of many reproductive 69 barriers (Brar and Khush 1997; Sitch 1990;Draft Khush and Brar 1992). Therefore, a major portion of the 70 genetic richness from secondary and tertiary gene pools are still untapped (Zhu et al. 2007; Palmgren 71 et al. 2014). 72 Monosomic alien addition lines (MAALs) is considered as one of the successful technique for 73 transferring useful traits from distantly related species to cultivated rice. Several useful genes have 74 been transferred from distantly related genome to cultivated rice, for example, brown planthopper 75 (BPH) and white backed planthopper (WBPH) from O. officinalis (Jena and Khush 1990), Bacterial 76 blight and blast resistance genes from O. minuta (Amante-Bordeos et al. 1992), BPH resistance 77 genes, earliness, awn length, days to flowering, and bacterial blight from O. australiensis (Ishii et al. 78 1994; Multani et al. 1994), yield components, bacterial blight and lodging resistance from O. 79 latifolia (Multani et al. 2003; Angeles-Shim et al. 2014), Blast, BPH and green leafhopper (GLH) 80 resistance genes from O. rhizomatis (Hechanova et al. 2018). Monosomic alien addition lines 81 (MAALs) of O. punctata has also been produced for the identification and transfer of BPH, GLH, 82 blast, and bacterial blight resistance (Yasui and Iwata 1991; Jena et al. 2016). However, development 83 and characterization of MAAL is a daunting task and required considerable time and skills for sexual 3 https://mc06.manuscriptcentral.com/genome-pubs Genome Page 4 of 28 84 hybridization followed by embryo rescue, each time. Therefore, colchicine-induced chromosome 85 doubling of an interspecific hybrid is one of the important methods to restore fertility. 86 87 Development of synthetic amphiploid via colchicine-induced chromosome doubling is 88 another strategy to overcome pollen sterility of interspecific hybrids and eliminate the post-zygotic 89 barrier by improving chromosome pairing (Wulff and Moscou 2014; De Paula et al. 2017; Yi et al. 90 2015). Synthetic amphiploid contains an additional set of genome exhibit high flexibility for 91 hybridization and hence, act as a bridge and genetic buffer in the distant hybridization (Cai et al. 92 2001). Furthermore, these new germplasm resources of rice containing an additional set of alien 93 genome can be used to study genetic relationships among different genomes or in research on rice 94 evolution (Kim et al. 2007). 95 The African wild rice, O. punctata Kotschy ex Steud. is a member of the O. 96 officinalis complex having two genomic configurations,Draft diploid (BB) and tetraploid (BBCC) (Jena et 97 al. 2016). Wild species, O. punctata has the untouched genetic variation that might be utilize for crop 98 improvement. In our previous report, the O. punctata acc. IRGC105137 was found resistant to 99 the Xanthomonas pathotypes, PbXo-7, PbXo-8 and PbXo-10 of bacterial leaf blight (Vikal et al. 100 2007; Neelam et al. 2016) and to the most virulent BPH biotype 4 of the Indian subcontinent (Sarao et 101 al. 2016). Though, few reports have been found for identification and exploitation of the gene 102 from O. punctata to cultivated rice (Yasui and Iwata 1991; Wang et al. 2013; Zhang et al. 2013; Jena 103 et al. 2016). 104 In the present study, we developed and charecterized a synthetic amphiploid (AABB) derived 105 from a cross between O. punctata IRGC105137 and O. sativa cv. Punjab Rice 122 (PR 122). Later, 106 this amphiploid was backcrossed with PR122 to generate addition/subtitution lines and phenotypically 107 evaluated them for the presence of agronomically important traits. 108 109 Material and methods 110 Plant materials 4 https://mc06.manuscriptcentral.com/genome-pubs Page 5 of 28 Genome 111 The diploid accession of O. punctata IRGC105137 (BB, 2n=2x=24) was maintained at the School of 112 Agricultural Biotechnology, Punjab Agricultural University (PAU), Ludhiana, Punjab, India. A cross 113 was attempted between O. punctata IRGC105137 and cultivated rice, O. sativa cv. Punjab Rice 122 114 (PR122, AA, 2n= 2x= 24). The synthetic amphiploid rice (AABB, 2n = 4X = 48) was developed from 115 the chromosome doubling of F1 hybrids. The diploid F1 hybrid (AB, 2n=2x=24) of O. punctata 116 IRGC105137 and PR122 and synthetic amphiploid (AABB, 2n=4x=48) were used for 117 cytomorphological studies and for the development of backcross derivatives (Figure S1). 118 Crosses and embryo rescue 119 The cultivated rice variety, PR122 was used as the female parent and the pollens of O. punctata were 120 dusted on emasculated florets of PR122. After 24 hrs of pollination, hormone treatment with GA3 (75 121 ppm) was done to control shattering. After twelve days of pollination, the immature hybrid embryos 122 were excised and cultured on half-strength Murashige and Skoog (MS) media. The cultured embryos 123 were incubated in the dark under controlledDraft temperature conditions i.e. 25±1oC. When embryos 124 germinated, the culture tubes were shifted to the light in the incubation room with 16-18 hrs of the 125 light period. After the establishment of plantlets with sufficient roots, they were transferred for 126 hardening in coco-peat. Fully hardened plants were then transplanted to the field after proper 127 establishment. 128 Colchicine-induced chromosome doubling 129 After 30 days of transplantation, the detached F1 seedlings were treated with 0.2% aqueous solution of 130 colchicine and 2% DMSO for 5 hrs. followed by washing with running tap water for overnight. 131 Seedlings were transferred to the field after treatment.
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