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.
132 Cytological studies
133 To study chromosome number and chromosomal behaviour in meiosis of diploid hybrids and
134 amphiploid, immature heads were collected in the early morning ( between 6 am to 8 am) and kept in
135 Corney’s solution (3 Alcohol: 1 glacial acetic acid) with the trace of ferric sulfate for 24 hrs. Then it is
136 stored in 70% ethanol at 4ºC until it is used.
137 Morphological data
5 https://mc06.manuscriptcentral.com/genome-pubs Genome Page 6 of 28
138 The morphological data such as plant height, tillers per plant, flag leaf length, flag leaf width,
139 spikelets per panicle, chlorophyll content, panicle length and days to heading were taken from F1,
140 amphiploid (A1 generation) and PR122.
141 Flow cytometric analysis
142 Analysis of nuclear DNA content of F1 and amphiploid were performed using Accuri C6 flow
143 cytometer (BD Biosciences) as per the instruction is given by the manufacturer’s manual. The diploid
144 F1 was taken as standard in this study. Tender leaves were placed in a plastic Petri plate and chopped
145 finely in 1ml of extraction buffer containing DNA fluorochrome solution and PI-RNase (PI- 50 μg/ml
146 in 0.1 % sodium citrate + 0.05% of NP 40 and DNase free RNase 2μg/ml) using razor blade in order
147 to isolate nuclei. Homogenate was then filtered through a 42 mm nylon mesh into a labeled sample
148 tube. First, both standard and samples were run separately and then mixed both sample in a single
149 tube to compare the DNA content. The analysis was based on light- scatter and fluorescence signal
150 produced from laser illumination at 488Draft nm. A bi-parametric contour plots and histogram were
151 prepared using BD CFlow® software against FL2- A versus FL3- A and a univariate histogram of
152 FL2- A.
153
154 Leaf anatomy of synthetic amphiploid
155 For studying leaf anatomy, widest part of the flag leaves of 3 independent rice plants from O.
156 punctata, PR122 and synthetic amphiploid were taken for sectioning. Fine vertical sections were
157 made manually. Samples were cleaned using a serial dilution of 30%, 50%, 70%, 90%, and 95%
158 ethanol and stained with safranin and toluidine blue. For mesophyll and bundle sheath cell study,
159 sections were kept overnight in 95% ethanol. Images were captured using a bright field microscope
160 (LMI, UK). Mesophyll cell measurement was carried out using Fiji-ImageJ software.
161 Results
162 Crosses and interspecific F1 hybrids
163 A cross was made using O. punctata IRGC105137 and PR122, cultivated rice to produce F1. Out of
164 75 embryos, 11 were germinated and of which only two plants were survived after hardening. The
6 https://mc06.manuscriptcentral.com/genome-pubs Page 7 of 28 Genome
165 presence of purple stigma, long awns, seed shattering and thick stem resembled that of O. punctata in
166 F1 hybrids. From two F1 plants, we generated 415 plants through clonal propagation by detaching
167 tillers and were planted at different locations in the field (Figure 1A). Hybrid plants were maintained
168 by ratooning every season at Punjab Agriculture University, Ludhiana, India.
169 Colchicine-induced chromosome doubling
170 A total of 69 clonally propagated F1 seedlings were treated with colchicine, of which 46 seedlings
171 were survived after treatment. Partially fertile pollen grains were found in the survived plants. Around
172 228 bulk seeds were collected from chromosome doubled plants. From 228 seeds, 39 plants
173 (Hereafter we referred as amphiploid 1 (A1) generation) were germinated in Kharif season 2014 and
174 grown in two replication by splitting tillers of 40 days old seedling. Pollen fertility was found 50%
175 (Figure 1B). Due to the high level of shattering, pollens were dusted in each replication separately by
176 clipping method in the field and allow it to open pollinate as well. Seeds were collected at regular
177 intervals of 5-8 days till the plants attainedDraft physiological maturity.
178 Cytological studies and meiotic behaviour of F1 hybrids and amphiploid
179 Pollen mother cells (PMCs) from immature heads of F1 hybrids and amphiploid were cytologically
180 analyzed. Of the 20 PMCs of F1 hybrids, an average of 19-20 univalents and 1-3 bivalent was
181 observed. In synthetic amphiploid, normal bivalent (24II) observed during meiosis (Figure 1C).
182 Morphology of the synthetic amphiploids
183 The “Giga” characters such as culm thickness, plant height, wider and dark green leaf, longer flag
184 leaf, etc were found in amphiploids. Normal seed set was observed in amphiploids. Seeds were found
185 bolder than both of their diploid parents. Amphiploids found taller than its hybrids and diploid
186 cultivated parent, PR122. Awns were longer than F1s and O. punctata. Days to heading in both were
187 similar which represent earliness than the cultivated parent. Anthocyanin pigmentation in awns of
188 selected synthetic amphiploid was also observed. A high degree of shattering was observed in
189 amphiploid while backcrossing with cultivated rice as well. The amphiploids showed significant
190 improvement in root morphology than both of the parents (Figure 2). A significant decrease in tillers
191 number per plant and spikelets per panicle was seen in amphiploids as compared to the F1 and PR122.
7 https://mc06.manuscriptcentral.com/genome-pubs Genome Page 8 of 28
192 However, no changes were observed in flag leaf width and SPAD analysis conducted for chlorophyll
193 content (Table 1).
194 Flow cytometric analysis
195 Analysis of nuclear DNA content produced sharp DNA peaks with relatively low coefficients of
196 variation (CV). The bi-parametric distribution (FL2-A vs FL3-A) of a Propidium Iodide (PI) stained
197 homogenate prepared from F1 hybrid tissue (Figure S2). The mean fluorescent value of the peak
198 position, 28871.37 with 3.70% CV was observed for diploid content (2C) of F1 hybrid. Similarly,
199 homogenate prepared from synthetic amphiploid tissue, the mean fluorescent value observed was
200 62843.37 with CV 2.88% (Figure 2E and 2F). In order to compare DNA content of diploid and
201 synthetic amphiploid, homogenate prepared from both tissues were mixed in a single tube and the
202 mean of peak position was recorded 29323.31 and 61478.23 for F1 hybrid and synthetic amphiploid
203 respectively which is almost similar as noted earlier (Figure 2G). The CVs were 3.85% for 2C and
204 2.82% for 4C. Draft
205 Leaf anatomy of synthetic amphiploids
206 Synthetic amphidiploids showed remarkable differences in morphological and anatomical features as
207 compared to both the parents. Mesophyll cells in the leaves of amphidiploid plants appear to be
208 significantly larger than both of its parents. Larger mesophyll cells in amphidiploids may also
209 correspond to a higher amount of chlorophyll in the leaves of amphidiploids (Figure 3A). Bundle
210 sheath cells around the major vein of amphidiploids were larger than both of the parents in mature
211 leaves (Figure 3B, 3C). Taken together, larger mesophyll cell size and bundle sheath cell size may
212 contribute to the increased biomass of the amphidiploid plants compared to both the parents.
213 Identification of aneuploids
214 Seeds collected from the A1 generation were planted in Kharif season 2015. Around 8500 plants were
215 grown and maintained over the different location in the field. Since it is expected to be a mixture of
216 crossed and self-seeds, each individual progeny row was carefully examined with the expectation to
217 identify and isolate aneuploid lines including triploid plants. Twenty plants have identified in the field
218 which was further characterized cytologically and backcrossed with PR122 (Figure 4A-C).
8 https://mc06.manuscriptcentral.com/genome-pubs Page 9 of 28 Genome
219 Cytomorphological characterization of aneuploids
220 Triploids are generally weak in plant type and sterile. With these facts, we identified 16 weak plants
221 and four healthy plants. At later stages, four healthy plants were found completely sterile and the plant
222 type was more or less similar to a hybrid plant. Cytological observations of pollen mother cells
223 revealed that the chromosome numbers of sterile plants were 23-25 with 3-4 bivalents (Figure 4D-F).
224 These observations are indicated towards the improper meiotic segregation of chromosome during
225 cell division.
226 High grain number trait in backcross progeny
227 Attempts were made to backcross amphiploid with the recurrent parent, PR122 and succeeded to
228 produce thirty-nine backcross progenies. Few of them possess high grain number than PR122 (Figure
229 5). Cytological examination, other useful agronomic traits of these backcross progenies is underway.
230 Data for grain number of selected backcross progeny was given in Table 2.
231 Draft
232 Discussion
233 Synthetic amphiploid offers several advantages in crop improvement such as gene discovery,
234 transferring genes from distant genome to cultivated gene pool as a bridge, studying the ancient
235 evolutionary processes and comparative genomics studies (Cai et al. 2001; Zhang et al. 2014).
236 Synthetic amphiploids were produced in different crops for the identification of useful abiotic and
237 biotic stress resistance genes along with quality traits, such as in rice (Zhang et al. 2014; Yi et al.
238 2015), wheat (Peng et al. 2003; Jauhar et al. 2004; Neelam et al. 2013), Cotton (Chen et al. 2015; Liu
239 et al. 2015), groundnut (Burrow et al. 2001; Mallikarjuna et al. 2010; Favero et al. 2015; Michelotto
240 et al. 2017), oat (Ladizinsky 2000; Ueno and Morikawa 2007), Brassica (Song et al. 1993; Banga et
241 al. 2003). In this study, we characterised a synthetic amphiploid between O. punctata and O. sativa
242 and their backcrossed derivatives.
243 Morphophysiological characteristic of synthetic amphiploid
244 A significantly reduced tiller number and spikelets per panicle were observed in synthetic
245 amphiploids. The most probable explanation for the drastic phenotypic variation may due to
9 https://mc06.manuscriptcentral.com/genome-pubs Genome Page 10 of 28
246 chromosome rearrangements and chromosomal abnormalities during the abrupt doubling of the
247 genome (Ainouche et al. 2009; Mestiri et al. 2010; Wu et al. 2018). Earlier, intermediate tiller
248 number and comparatively less grain number was observed in synthetic amphiploid than insterspecific
249 hybrid derived from O. sativa and O. latifolia cross (Yi et al. 2015). Heterosis observed for flag leaf
250 length, plant height and awns in F1s and amphiploids might be attributed to the epistatic nature of the
251 genes controlling these traits in O. punctata. A noteworthy increase in the size of mesophyll cells and
252 bundle sheath cells in synthetic amphiploid might be due to increased dosage of the chromosomes.
253 The ultimate effects of these improved traits over both the parents are obvious in backcrossed
254 progenies with higher grain number. The increased length of flag leaf was correlated with higher grain
255 yield and biomass accumulation by escalating canopy photosynthesis in rice by various workers
256 (Fujita et al. 2012; Fujita et al. 2013; Fabre et al. 2016). Several QTLs/ genes have been mapped to
257 uncover effect of shape and size of flag leaves for establishing source-sink relationship using QTL
258 mapping and mutant identification approachesDraft (Paul and Foyer 2001; Hu et al. 2010; Xiang et al.
259 2012; Zhang et al. 2015; Luo et al. 2013).
260 Homoeology of the A and B genome
261 In this report, hybrids produced from a cross between O. punctata and O. sativa showed a
262 high level of male sterility. In general, hybrids derived from wide hybridization often show a high
263 level of sterility (Sitch et al. 1989). The sterility might be due to lack of sufficient structural and
264 numerical homology between wild and cultivated chromosomes which resulted into the formation of
265 unbalanced gametes and further abortion of pollen grains (Mariam et al. 1996). Similar observations
266 were also reported in wide crosses derived O. punctata and cultivated species (Yasui and Iwata 1991;
267 Wang et al. 2013; Jena et al. 2016). In this study, we observed 19- 20 univalents and 1-3 bivalents per
268 cell in the PMCs of F1 hybrid. Earlier, Wang et al (2013) also showed 4.89–8.52 bivalents in the
269 PMCs of F1 hybrid derived from O. sativa and O. punctata cross. However, Jena et al (2016) observed
270 no bivalents in F1 hybrid derived from cross between O. sativa and O. punctata. Li et al (1962) and
271 Multani et al (1994) observed 0-8 bivalent in the interspecific hybrid. Presence of bivalents may be
272 explained by the fact of phylogenetically close relationship between A and B genome of rice (Ge et al.
273 2005; Nishikawa et al. 2005). Comparative physical mapping between O. sativa and O. punctata
10 https://mc06.manuscriptcentral.com/genome-pubs Page 11 of 28 Genome
274 genome by Kim et al (2007) concluded that chromosome 6 and 12 were the most diverged and
275 chromosome 3 was the most similar. Khush (2010) and Jena et al (2016) reported that the rice
276 chromosomes 4 and 11 have high transmission frequency based on trisomics and monosomic alien
277 addition line derived from O. punctata, respectively. We observed four sterile plants in the open
278 pollinated population of synthetic amphiploid and few backcross progeny attained tallness at
279 reproductive phase. Male sterility and tallness in trisomics of rice were controlled by genes on
280 chromosome 4 and 12, respectively (Khush et al. 1984) explaining the above mentioned phenomenon.
281 Introgression of useful traits from O. punctata in backcrossed derivatives
282 In our investigation, we obtained backcross derivatives with higher grain number as compared
283 to the recepient rice cultivars PR122, indicating the presence of distinctive genetic system in O.
284 punctata for the improvement for yield components traits. The presence of grain number traits have
285 also been identified in synthetic amphiploids generated in other crops such as wheat (Miko et al.
286 2015; Yan et al. 2017; Oneymaobi et al. 2018)Draft and brassica (Malek et al. 2012). The other remarkable
287 agronomically value added traits from O. punctata to the backcrossed progenies was earliness and
288 higher biomass. The days to heading of PR122 is ranges from 107 to 110 days whereas the backcross
289 progenies took only 75 to 90 days giving an advantage of almost one month over PR122. These lines
290 could be further utilize in breeding program for the develpoment of short duration ‘climate smart’
291 varieties and facilitate water saving in the era of climate change.
292
293 Conclusion
294 In this study, we developed a synthetic amphiploids by colchicine-induced chromosome doubling of
295 an interspecific hybrid derived from cross between O. punctata and O. sativa cv. PR122.
296 Chromosome number in PMCs and genomic content of the synthetic amphiploids was doubled than
297 diploid F1 hybrids confirmed by cytological examination and flow cytometric analysis. Distinct
298 alteration in phenotype due to colchicine-induced chromosome doubling in the synthetic amphiploids
299 were observed. Synthetic amphiploids could be an excellent germplasm resources to develop series
300 addition/substitution lines and can be utilized in the study of the dosage of genes, expression of
11 https://mc06.manuscriptcentral.com/genome-pubs Genome Page 12 of 28
301 reccesive traits, and identification of genes. Synthetic amphiploid can also be used as a bridge in
302 distant hybridization to transfer genes from unadopted wild relatives to cultivated gene pool.
303 Acknowledgement
304 The authors are thankful to the International Rice Research Institute, Philippines, Manila for sharing
305 wild species germplasm of rice. Author also thank the Indian Council of Agricultural Research
306 (ICAR), New Delhi funded project “Niche Area of Excellence in Wheat and Rice, PC 2198” for
307 providing financial grant.
308 Conflict of Interests
309 Author declare that they have no conflicts of interests
310 References
311 Ainouche, M.L., Fortune, P., Salmon, A., Parisod, C., Grandbastien, M.A., Fukunaga, K., Ricou, 312 M., and Misset, M.T. 2009. Hybridization,Draft polyploidy and invasion: lessons 313 from Spartina (Poaceae). Biol. Invasions, 11: 1159– 1173.
314 Amante-Bordeos, A., Sitch, L.A., Nelson, R., Dalmacio, R.D., Oliva, N.P., Aswidinnoor, H., and
315 Leung H. 1992. Transfer of bacterial blight and blast resistance from the tetraploid wild rice
316 Oryza minuta to cultivated rice, Oryza sativa. Theor. Appl. Genet. 84:345-354.
317 Angeles-shim, R.B., Vinarao, R.B., Marathi, B., and Jena, K.K. 2014. Molecular analysis of Oryza
318 latifolia Desv. (CCDD Genome) derived introgression lines and identification of value-added
319 traits for rice (O. sativa L.) improvement. J. Hered. 105:676-689.
320 Banga, S.S., Bhaskar, P.B., and Ahuja, I. 2003. Synthesis of intergeneric hybrids and establishment of
321 genomic affinity between Diplotaxis catholica and crop Brassica species. Theor. Appl.
322 Genet. 106: 1244–1247.
323 Brar, D.S., and Khush, G.S. 1997. Alien introgression in rice. Plant Mol. Biol. 35:35-47.
324 Burow, M.D., Simpson, C.E., Starr, J.L., and Paterson, A.H. 2001. Transmission genetics of
325 chromatin from a synthetic amphidiploid to cultivated peanut (Arachis hypogaea L.):
326 broadening the gene pool of a monophyletic polyploidy species. Genetics, 159: 823-37.
12 https://mc06.manuscriptcentral.com/genome-pubs Page 13 of 28 Genome
327 Cai, D.T., Yuan, L.P., and Lu, X.G. 2001. A new strategy of rice breeding in the 21st century.
328 Searching a new pathway of rice breeding by utilization of double heterosis of wide cross and
329 polyploidization. Acta Agron. Sin. 27:110-116.
330 De Paula, A.F., Dinato, N.B., Vigna, B.B.Z., Fávero, A.P. 2017. Recombinants from the crosses
331 between amphidiploid and cultivated peanut (Arachis hypogaea) for pest-resistance breeding
332 programs. PLoS One 12: e0175940. https://doi.org/10.1371/journal.pone.0175940.
333 Fabre, D., Adriani, D.E., Dingkuhn, M., Ishimaru, T., Punzalan, B., Lafarge, T., Clément-Vidal, A.,
334 and Luquet, D. 2016. The qTSN4 Effect on Flag Leaf Size, Photosynthesis and Panicle Size,
335 Benefits to Plant Grain Production in Rice, Depending on Light Availability. Front. Plant Sci.
336 7:623. https://doi.org/10.3389/fpls.2016.00623.
337 Fávero, A.P., Pádua, J.G., Costa, T.S., Gimenes, M.A., Godoy, I.J., Moretzsohn, M.C., and 338 Michelotto, M.D. 2015. New hybridsDraft from peanut (Arachis hypogaea L.) and synthetic 339 amphidiploid crosses show promise in increasing pest and disease tolerance. Genet. Mol. Res.
340 14: 16694-16703.
341 Fujita, D., Tagle, A.G., Ebron, L.A., Fukuta, Y., and Kobayashi, N. 2012. Characterization of near-
342 isogenic lines carrying QTL for high spikelet number with the genetic background of an indica
343 rice variety IR64 (Oryza sativa L.). Breed. Sci. 62:18-26.
344 Fujita, D., Trijatmiko, K.R., Tagle, A.G., Sapasap, M.V., Koide, Y., Sasaki, K., Tsakirpaloglou, N.,
345 Gannaban, R.B., Nishimura, T., Yanagihara, S., Fukuta, Y., Koshiba, T., Slamet-Loedin, I.H.,
346 Ishimaru, T., and Kobayashi, N. 2013. NAL1 allele from a rice landrace greatly increases yield
347 in modern indica cultivars. Proc. Natl. Acad. Sci. USA. 110:20431-6.
348 Ge, S., Guo Y.-L., and Zhu, Q.-H. 2005. Molecular phylogeny and divergence of the rice tribe
349 (Oryzeae), with special reference to the origin of the genus Oryza. In Rice Is Life: Scientific
350 Perspectives for the 21st Century. Edited by K. Toriyama, K. L. Heong and B. Hardy. IRRI,
351 Philippines. pp. 40–44.
13 https://mc06.manuscriptcentral.com/genome-pubs Genome Page 14 of 28
352 Hechanova, S.L., Prusty, M.R., Kim, SR., Ballesfin, L., Ramos, J., Prahalada, G.D., and Jena, K.K.
353 2018. Monosomic alien addition lines (MAALs) of Oryza rhizomatis in Oryza sativa:
354 production, cytology, alien trait introgression, molecular analysis and breeding application.
355 Theor. Appl. Genet. 131: 2197. https://doi.org/10.1007/s00122-018-3147-x.
356 Hu, J., Zhu, L., Zeng, D., Gao, Z., Guo, L., Fang, Y., Zhang, G., Dong, G., Yan, M., Liu, J., and Qian,
357 Q. 2010. Identification and characterization of NARROW AND ROLLED LEAF 1, a novel gene
358 regulating leaf morphology and plant architecture in rice. Plant Mol. Biol. 73:283-92.
359 Ishii, T., Brar, D.S., Multani, D.S., and Khush, G.S. 1994. Molecular tagging of genes for brown
360 planthopper resistance and earliness introgressed from Oryza australiensis into cultivated rice,
361 O. sativa. Genome, 37:217-221.
362 Jauhar, P.P., Doğramacı, M., and Peterson, T.S. 2004. Synthesis and cytological characterization of 363 trigeneric hybrids of durum wheat withDraft and without Ph1. Genome, 47: 1173-1181. 364 Jena, K. K., Ballesfn, M. L. E., and Vinarao, R. B. 2016. Development of Oryza sativa L. by Oryza
365 punctata Kotschy ex Steud. monosomic addition lines with high value traits by interspecifc
366 hybridization. Theor Appl Genet. 129:1873–1886. https://doi.org/10.1007/s00122-016-2745-8.
367 Jena, K.K. 2010. The species of the genus Oryza and transfer of useful genes from wild species into
368 cultivated rice, O. sativa. Breed. Sci. 60:518-523.
369 Jena, K.K., and Khush, G.S. 1990. Introgression of genes from Oryza officinalis Well ex Watt to
370 cultivated rice, O. sativa L. Theor. Appl. Genet. 80:737-745.
371 Khush, G.S., Singh, R.J., Sur, S.C., and Librojo, A.L. 1984. Primary trisomics of rice: origin,
372 morphology, cytology and use in linkage mapping. Genetics, 107: 141-163.
373 Khush, G.S. 1997. Origin, dispersal, cultivation and variation of rice. Plant Mol. Biol. 35:25-34.
374 Khush, G.S. 2010. Trisomics and alien addition lines in rice. Breed. Sci. 60:469–474.
375 Khush, G.S. and Brar, D.S. 1992. Overcoming the barriers in hybridization. Theor. Appl. Genet. 16:
376 47-61.
14 https://mc06.manuscriptcentral.com/genome-pubs Page 15 of 28 Genome
377 Kim, H.R., Miguel, P.S., Nelson, W., Collura, K., Wissotski, M., Walling, J.G., Kim, J.P.,
378 Jackson, S.A., Soderlund, C., Wing, R.A. 2007. Comparative physical mapping between Oryza
379 sativa (AA genome type) andO. punctata (BB genome type). Genetics, 176:379-390.
380 https://doi.org/10.1534/genetics.106.068783.
381 Ladizinsky, G. 2000. A synthetic hexaploid (2n=42) oat from the cross of Avena strigosa (2n=14)
382 and domesticated A. magna (2n=28). Euphytica, 116:231-235.
383 Li, H.W., Weng, T.S., Chen, C.C., Wang, W.H. 1962. Cytogenetical studies of Oryza sativa L. and its
384 related species, 2. A preliminary note on the interspecific hybrids within the section Sativa
385 Roschev. Bot. Bull. Acad. Sin. 3:209–219.
386 Liu, Q., Chen, Y., Chen, Y., Wang, Y., Chen, J., Zhang, T., and Zhou, B. 2015. A New Synthetic
387 Allotetraploid (A1A1G2G2) between Gossypium herbaceum and G. australe: Bridging for 388 simultaneously transferring favorableDraft genes from these two diploid species into upland cotton. 389 PLoS One 10: e0123209. https://doi.org/10.1371/journal.pone.0123209.
390 Luo, X., Ji, S.D., Yuan, P.R., Lee, H.S., Kim, D.M., Balkunde, S., Kang, J.W., and Ahn, S.N. 2013.
391 QTL mapping reveals a tight linkage between QTLs for grain weight and panicle spikelet
392 number in rice. Rice. 6:33.
393 Malek, M.A., Ismail, M.R., Rafii, M.Y., and Rahman, M. 2012. Synthetic Brassica napus L.:
394 development and studies on morphological characters, yield attributes, and yield. Sci. World J.
395 https://doi.org/10.1100/2012/416901.
396 Mallikarjuna, N., Senthilvel, S., and Hoisington, D. 2010. Development of new sources of tetraploid
397 Arachis to broaden the genetic base of cultivated groundnut (Arachis hypogaea L.). Genet.
398 Resour. Crop Evol. https://doi.org/10.1007/s10722-010-9627-8.
399 Mariam, A.L., Zakri, A.H., Mahani, M.C., and Normah, M.N. 1996. Interspecific hybridization of
400 cultivated rice, Oryza sativa L. with the wild rice, O. minuta Presl. Theor. Appl. Genet. 93:664-
401 671. https://doi.org/10.1007/bf00224060.
15 https://mc06.manuscriptcentral.com/genome-pubs Genome Page 16 of 28
402 Mestiri, I., Chague, V., Tanguy, A.-M., Huneau, C., Huteau, V., Belcram, H., Coriton, O., Chalhoub,
403 B., and Jahier, J. 2010. Newly synthesized wheat allohexaploids display progenitor-dependant
404 meiotic stability and aneuploidy but structural genomic additivity. New Phytol. 186: 86–101.
405 Michelotto, M.D., de Godoy, I.J., Pirotta, M.Z., dos Santos, J.F., Finoto, E.L., and Pereira Fávero, A.
406 2017. Resistance to thrips (Enneothrips flavens) in wild and amphidiploid Arachis
407 species. PLoS One 12:e0176811. https://doi.org/10.1371/journal.pone.0176811.
408 Mikó, P., Megyeri, M., Farkas, A., Molnár, I., and Molnár‐Láng, M. 2015. Molecular cytogenetic
409 identification and phenotypic description of a new synthetic amphiploid, Triticum timococcum
410 (AtAtGGAmAm). Genet. Resour. Crop Evol. 62: 55‐66.
411 Multani, D.S., Jena, K.K., Brar, D.S., Reyes, B.G., Angeles, E.R., and Khush, G.S. 1994.
412 Development of monosomic alien addition lines and introgression of genes from Oryza
413 australiensis Domin. to cultivated riceDraft O. sativa L. Theor. Appl. Genet. 88:102-109.
414 Multani, D.S., Khush, G.S., Reyes, B.G., and Brar, D.S. 2003. Alien genes introgression and
415 development of monosomic alien addition lines from Oryza latifolia Desv. to rice, Oryza sativa
416 L. Theor. Appl. Genet. 107:395-405.
417 Neelam, K., Lore, J.S., Kaur, K., Pathania, S., Kumar, K., Sahi, G. K., Mangat, G.S., and Singh, K.
418 2016. Identifcation of resistance sources in wild species of rice against two recently evolved
419 pathotypes of Xanthomonas oryzae pv oryzae. Plant Genet. Resour.
420 https://doi.org/10.1017/s1479262116000149.
421 Neelam, K., Rawat, N., Tiwari, V.K., Ghandhi, N., Patokar, C.A., Kumar, S., Tripathi, S.K.,
422 Randhawa, G. S., Prasad, R., and Dhaliwal, H.S. 2013. Development and molecular
423 characterization of wheat- 'Aegilops longissima' derivatives with high grain micronutrients.
424 Aus. J. Crop. Sci. 7: 508.
425 Nishikawa, T., Vaughan, D.A., and Kadowaki, K. 2005. Phylogenetic analysis species, based on
426 simple sequence repeats and their flanking nucleotide sequences from the mitochondrial and
427 chloroplast genomes. Theor. Appl. Genet. 110:696–705.
16 https://mc06.manuscriptcentral.com/genome-pubs Page 17 of 28 Genome
428 Onyemaobi, I., Ayalew, H., Liu, H., Siddique, K.H.M., and Yan, G. 2018. Identification and
429 validation of a major chromosome region for high grain number per spike under meiotic stage
430 water stress in wheat (Triticum aestivum L.). PLoS One, 13: e0194075.
431 https://doi.org/10.1371/journal.pone.0194075.
432 Palmgren, M.G., Edenbrandt, A.K., Vedel, S.E., Andersen, M.M., Landes, X., Østerberg, J.T., Falhof,
433 J., Olsen, L.I., Christensen, S.B., Sandøe, P., Gamborg, C., Kappel, K., Thorsen, B.J., and Pagh,
434 P. 2015. Are we ready for back-to-nature crop breeding? Trends Plant Sci. 20:155-64.
435 Paul, M.J., and Foyer, C.H. 2001. Sink regulation of photosynthesis. J. Exp. Bot. 52:1383-400.
436 Peng, J., Ronin, Y., Fahima, T., Röder, M.S., Li, Y., Nevo, E., and Korol, A. 2003. Domestication
437 quantitative trait loci in Triticum dicoccoides, the progenitor of wheat. Proc. Natl. Acad.
438 Sci. 100:2489–2494.
439 Sarao, P.S., Sahi, G.K., Neelam, K. etDraft al. 2016. Donors for resistance to brown planthopper 440 Nilaparvata lugens (Stål) from wild rice species. Rice Sci. 23:219–224.
441 https://doi.org/doi:10.1016/j.rsci.2016.06.005.
442 Sitch, L. A. 1990. Incompatibility barriers operating in crosses of Oryza sativa with related species
443 and genera. In Genetic Manipulation in Plant Improvement II. Edited by J. P. Gustafson.
444 Plenum Press, New York, pp. 77-94.
445 Sitch, L.A., Romero, G.O., Dalmacio, R.D. 1989. Pre-fertilization incompatibility barriers in
446 interspecific and intergeneric crosses involving Oryza sativa. Int. Rice Res. Newsl. 14:5-6.
447 Song, K.M., Tang, K., and Osborn, T.C. 1993. Development of synthetic Brassica amphidiploids by
448 reciprocal hybidization and comparision to natural amphidiploids. Theor. Appl. Genet. 86:811-
449 21.
450 Tanksley, S.D., and McCouch, S.R. 1997. Seed banks and molecular maps: Unlocking genetic
451 potential from the wild. Science 277:1063-1066.
452 Ueno, M., and Morikawa, T. 2007. Productionof synthetic polyploid oats and detection of C genome
453 rearrangements by GISH and FISH. Breed. Sci. 57: 339-343.
17 https://mc06.manuscriptcentral.com/genome-pubs Genome Page 18 of 28
454 Vaughan, D.A. 1989. The genus Oryza L: Current status of taxonomy. IRRI Research Paper Series
455 138. Manila: IRRI.
456 Vikal, Y., Das, A., Patra, B., Goel, R.K., Sidhu, J.S., and Singh, K. 2007. Identifcation of new sources
457 of bacterial blight (Xanthomonas oryzae pv.oryzae) resistance in wild Oryza species and O.
458 glaberrima. Plant Genet. Resour. 5:108–112.
459 Wang, A.Y., Zhang, X.H., Yang, C.H., Song, Z.J., Du, C.Q., Chen, D. L., He, Y., and Cai, D.T. 2013.
460 Development and characterization of synthetic amphiploid (AABB) between Oryza sativa and
461 Oryza punctata. Euphytica. 89:1-8.
462 Wu, Y., Sun, Y., Sun, S., Li, G., Wang, J., Wang. B., Lin, X., Huang, M., Gong, Z., Sanguinet,
463 K. A., Zhang, Z., Liu, B. 2018. Aneuploidization under segmental allotetraploidy in rice and its
464 phenotypic manifestation. Theor. Appl. Genet. 131: 1273. https://doi.org/10.1007/s00122-
465 018-3077-7. Draft
466 Wulff, B.B.H., Moscou, M.J. 2014. Strategies for transferring resistance into wheat: from wide
467 crosses to GM cassettes. Front. Plant Sci. 5:692. https://doi.org/10.3389/fpls.2014.00692.
468 Xiang, J.J., Zhang, G.H., Qian, Q., and Xue, H.W. 2012. Semi-rolled leaf1 encodes a putative
469 glycosylphosphatidylinositol-anchored protein and modulates rice leaf rolling by regulating the
470 formation of bulliform cells. Plant Physiol. 159:1488-500.
471 Yan, L., Liang, F., Xu, H., Zhang, X., Zhai, H., Sun, Q., and Ni, Z. 2017. Identification of QTL for
472 Grain Size and Shape on the D Genome of Natural and Synthetic Allohexaploid Wheats with
473 Near-Identical AABB Genomes. Front. Plant Sci. 8:1705.
474 https://doi.org/10.3389/fpls.2017.01705.
475 Yasui, H., and lwata, N. 1991. Production of monosomic alien addition lines of Oryza sativa
476 having a single O. punctata chromosome. In Rice genetics II. International Rice Research
477 Institute, P.O. Box 933, Manila, Philippines, pp. 147-155.
18 https://mc06.manuscriptcentral.com/genome-pubs Page 19 of 28 Genome
478 Yi, C., Wang, M., Jiang, W., Wang, D., Cheng, X., Wang, Y., Zhou, Y., Liang, G., and Gu, M. 2015.
479 Development and characterization of synthetic amphiploids of Oryza sativa and Oryza latifolia.
480 Sci. Bull. 60:2059–2062. https://doi.org/10.1007/s11434-015-0944-3.
481 Zhang, B., Ye, W., Ren, D., Tian, P., Peng, Y., Gao, Y., Ruan, B., Wang, L., Zhang, G., Guo, L.,
482 Qian, Q., and Gao, Z. 2015. Genetic analysis of flag leaf size and candidate genes
483 determination of a major QTL for flag leaf width in rice. Rice, 8:39.
484 Zhang, X., Wang, A., Du, C., Song, Z., Wang, W., He, Y., Cai, D. 2014. An efficient method of
485 developing synthetic allopolyploid rice (Oryza spp.). Genet. Resour. Crop Evol.
486 https://doi.org/10.1007/s10722-013-0075-0.
487 Zhang, X., Wang, W., Jin, J., Liao, S., Liu, X., Song, Z., and Cai, D. 2013. Cytomorphological
488 Characterization of the Backcross Progeny of Synthetic Amphiploid Rice (AABB) and
489 Tetraploid Oryza sativa (AAAA). J. Agri. Sci. 5:30-37.
490 Zhu, Q., Zheng, X., Luo, .J, Gaut, B.S., andDraft Ge, S. 2007. Multilocus analysis of nucleotide variation
491 of Oryza sativa and its wild relatives: severe bottleneck during domestication of rice. Mol. Biol.
492 Evol. 24:875-88.
19 https://mc06.manuscriptcentral.com/genome-pubs Genome Page 20 of 28
493 Table 1: Morphological comparison of F1, amphiploids (A1 generation) and PR122 for various agronomic traits
Flag leaf Flag leaf Panicle Days to Presence Pericarp Panicle Chlorophyll Plant height Tillers per widtha length Spiklets/Panicle Length heading of awns type content (in cm) plants (in cm) (in cm) (in cm) F1 2.90±0.11 31.75±3.69 139.15±10.73 47.345±2.59 105.88±1.96 128.13±24.57 27.84±1.80 62±6.30 Awned Red Compact
Amphiploids 2.99±0.21 43.9±5.13 91.20±9.80 48.365±2.53 129.13±6.20 35.25±6.14 28.60±2.30 61±4.39 Awned Red Medium
PR122 2.89±1.02 29.73±2.31 147.31±4.13 43.217±1.33 98.98±3.13 89.23±1.30 25.80±1.92 85±3.69 Awnless White Compact
494
495 aNumber of plants (n) analyzed was 20 except for plant height and tillers per plant which was n=8 496 Draft
20 https://mc06.manuscriptcentral.com/genome-pubs Page 21 of 28 Genome
497 Table 2: Grain number and heading date of selected backcross progeny
% decrease % Days to in heading Grain increase Pedigree Backcross Progeny heading## number/ date in grain Panicle number#
6109-1/PR122 82 190 17.4 25.45 6109-4/PR122 79 280 43.9 28.18 BC2F1[PR122/O.punctata 6110-1/PR122 85 170 7.7 IRGC105137 (amphiploid)//*2PR122] 22.73 21.5 6110-2/PR122 87 20.91 200 6110-3/PR122 75 170 7.7 31.82 6111-1/PR122 80 250 37.2 27.27 6111-2/PR122 82 178 11.8 25.45 6112-3/PR122 90 179 12.3 Draft 18.18 6112-4/PR122 88 170 7.6 20.00 PR122 110 157 498
499 # Percent increase of grain number was calculated on the basis of grain number of PR122 (157 grains/
500 panicle)
501 ## The mean heading date of PR122 was 110 days.
502
503
504
505
506
507
508
509
510
511
21 https://mc06.manuscriptcentral.com/genome-pubs Genome Page 22 of 28
512
513 Figure captions
514
515 Figure 1: Field view of F1 and synthetic amphiploid (A), stained pollen grains of F1 and synthetic
516 amphiploid (B), chromosomal behaviour during meiosis in the PMCs of F1 and synthetic amphiploid
517 (C).
518
519 Figure 2: Characteristics of the synthetic amphiploid. (A) Spikelet shattering in synthetic amphiploid,
520 (B) Anthocyaning pigmentation in awns of the synthetic amphiploid, (C), Auxillary root formation
521 above the crown region in synthetic amphiploid, red arrows indicate the auxillary roots (D) Seed
522 morphology of O. punctata IRGC105137 (1), O. sativa cv. PR122 (2), F1 (3), and synthetic
523 amphiploid (4), (E-G) Flow cytometric analysis of genome content of F , Synthetic amphiploid and Draft 1 524 mixture of both F1 and synthetic amphiploid.
525
526 Figure 3: Leaf anatomy of mesophyll cells of flag leaf (A), bundle sheath cells of fully expanded
527 vegetative leaf (B), and bundle sheath cells of flag leaf (C). Red arrows represent the bundle sheath
528 cells
529
530 Figure 4: Morphological features of selected aneuploids in the field (A-C) and segregation of
531 chromosomes during meiosis (D-F).
532
533 Figure 5: High grain number plants identified in the backcross progenies (BC) of cross between
534 synthetic amphiploid and PR122. (A) Panicles of backcross progenies with comparatively higher grain
535 number than recurrent parent, (B) PR122
536
537
538
539
22 https://mc06.manuscriptcentral.com/genome-pubs Page 23 of 28 Genome
540 Captions for supplementary figure
541
542 Figure S1 Schematic representation for production of synthetic amphiploids
543
544 Figure S2 Bi-parametric contour plot showing distribution (FL2-A vs FL3-A) of Propidium Iodide
545 (PI) stained homogenate prepared from Amphiploid, F1 hybrid and mixture of F1 hybrid tissues and 546 amphiploid 547
Draft
23 https://mc06.manuscriptcentral.com/genome-pubs Genome Page 24 of 28
Draft
Figure 1: Field view of F1 and synthetic amphiploid (A), stained pollen grains of F1 and synthetic amphiploid (B), chromosomal behavior during meiosis in the PMCs of F1 and synthetic amphiploid (C).
https://mc06.manuscriptcentral.com/genome-pubs Page 25 of 28 Genome
Figure 2: Characteristics of the synthetic amphiploid. (A) Spikelet shattering in synthetic amphiploid, (B) Anthocyaning pigmentation in awns of theDraft synthetic amphiploid, (C), Auxillary root formation above the crown region in synthetic amphiploid, red arrows indicate the auxillary roots (D) Seed morphology of O. punctata IRGC105137 (1), O. sativa cv. PR122 (2), F1 (3), and synthetic amphiploid (4), (E-G) Flow cytometric analysis of genome content of F1, Synthetic amphiploid and mixture of both F1 and synthetic amphiploid.
https://mc06.manuscriptcentral.com/genome-pubs Genome Page 26 of 28
Draft
Figure 3: Leaf anatomy of mesophyll cells of flag leaf (A), bundle sheath cells of a fully expanded vegetative leaf (B), and bundle sheath cells of flag leaf (C). Red arrows represent the bundle sheath cells
https://mc06.manuscriptcentral.com/genome-pubs Page 27 of 28 Genome
Draft
Figure 4: Morphological features of selected aneuploids in the field (A-C) and segregation of chromosomes during meiosis (D-F).
https://mc06.manuscriptcentral.com/genome-pubs Genome Page 28 of 28
Draft
Figure 5: High grain number plants identified in the backcross progenies (BC) of a cross between synthetic amphiploid and PR122. (A) Panicles of backcross progenies with comparatively higher grain number than the recurrent parent, PR122 (B).
https://mc06.manuscriptcentral.com/genome-pubs