Cytomorphological Characterization of the Backcross Progeny of Synthetic Amphiploid Rice ( AABB ) and Tetraploid Oryza sativa ( AAAA )

DCW008, a synthetic allopolyploid rice (2n = 4X = 48, AABB), was obtained from chromosome doubling of interspecific hybrids between Oryza sativa (2n = 24, AA) and O. punctata (2n = 24, BB). The F1 and backcross (BC1) hybrids were produced by crossing DCW008 as a female parent with a high seed set tetraploid rice Sg99012 4X (2n = 4X = 48, AAAA). BC1F1 and BC1F2 overcame many of the wild-type traits of DCW008; they had yellow-hulled grains, seed set ranged from 0% to 71.31% and the grain morphology was similar to that of cultivated rice. Variable numbers of chromosomes were observed in pollen mother cells (PMCs) from the BC1 plant. Genomic in situ hybridization (GISH) revealed that the majority of somatic cells and PMCs contained six chromosomes of O. punctata with fragment recombination observed in two of them. The backcross selection method employed in this study to generate allopolyploid progeny provides a reliable way of transferring useful genes from wild species into cultivated rice.


Introduction
The genus Oryza is composed of more than 20 species, represented cytogenetically by ten genome groups, including two cultivated species, O. sativa L. and O. glaberrima Steud (Li et al., 2001;Bao et al., 2006).Abundant wild species of Oryza provide extremely valuable genetic resources to broaden the genetic background of cultivated rice (Khush, 1977).Many researchers have, for a long time, tried to use these wild resources but reproductive barriers, especially at the diploid level, make it difficult to cross and transfer useful genes from wild rice to cultivated rice.However, allopolyploids, which have multiple chromosome sets, exhibit high plasticity since they have the evolutionary advantage of possessing additional genetic materials for growth and adaptation.Hence, they may play an important role in the distant hybridization and promote the exploitation of useful genes from wild species.Allopolyploidy has proven very useful in the utilization of genetic resources to increase yields in many crop species (Albertin et al., 2006;Goncharov et al., 2007;Flagel et al., 2008).Furthermore, allopolyploid rice is a versatile material that can be used to study relationships between different genome groups or in research on rice evolution.It can also be used as a bridge for transferring desirable genes into cultivated rice through gene introgression (Cai et al., 2001).This study utilized the synthetic allopolyploid rice DCW008 (AABB) (created by our laboratory and reported by Wang et al. 2013), which exhibits good fertility and advantageous agronomic traits.DCW008 was crossed and backcrossed with the high seed set tetraploid rice Sg99012-4X (AAAA) as a male parent to obtain the backcross derivatives.The genomic components and cytological characteristics of the BC 1 hybrids were studied using GISH.Grain yield and other major agronomic traits of F 1 , BC 1 F 1 and BC 1 F 2 were also investigated.

Crosses and Backcrosses
DCW008 was used as the female parent in crosses with Sg99012-4X.Hybrid plants were generated through embryo rescue according to the procedure described by Wang et al. (2013).First backcross generations (BC 1 ) were generated using Sg99012-4X as a recurrent male parent.The progeny were also produced through embryo rescue.The method for the generation of materials is shown in Figure 1.

Cytological Observations and GISH
Root tips from F 1 and backcross progeny were used for the determination of chromosome numbers.Directly fixed young inflorescences were used for meiotic behavior observation (Li et al., 1995).Pollen fertility was determined as the percentage of pollen grains stained with 0.2% fluorescein diacetate (FDA).
For GISH, root tip cells and selected anthers with pollen mother cells (PMCs) at suitable stages were digested for around 4 h in an enzyme mixture containing 2% cellulase and 2% pectinase.Chromosome preparation mainly followed the method described by Yan et al. (1998) with some modifications.Total genomic DNA was extracted from young leaves using the hexadecyltrimethylammonium bromide (CTAB) method.The DNA of O. punctata was fluorescently labeled with bio-11-dUTP using nick translation and used as the probe.The genomic DNA from Sg99012-4X was sheared by autoclaving for 5 min and used as the block.In situ hybridization was carried out according to the method of Leitch et al. (1990).

Morphological Observations
The key morphological traits of plant height, spike number and length, grain length and width, awn length, shattering trait, seed color and seed set were investigated.Recording methods and standards were set according to the protocols of Gai (1996).

F 1 Hybrids and Backcross Progeny
Seed set in the DCW008 × Sg99012-4X cross was 11.73% and the germination of rescued embryos was 61.90%.Twelve F 1 hybrid plants were obtained in total.The seed set upon backcrossing the F 1 hybrids with the recurrent female parent ranged from 4.38% to 6.25% across different years.A total of 31 BC 1 F 1 embryos were rescued, but only 10 germinated with a mean germination frequency of 34.10%.Six BC 1 F 1 plants survived in total (Table 1

Morphology of F 1 Plants and Backcross Progeny
The majority of the F 1 plants exhibited matroclinous morphology.All were of tall stature and tillering (Figure 2A).They had grain-shattering traits, long red awns, purple stigma and black hulled-grains.F 1 plants had no seed set after selfing although immature embryos could occasionally be observed.Compared with F 1 plants, the BC 1 plants' morphology was more similar to that of Sg99012-4X.FDA staining indicated most of the pollen was fertile (Figure 2B).Anthers were normal and full (Figure 2C) and the seed setting rate of the BC 1 F 1 plants ranged from 6.90% to 21.33% (Table 2).The shattering traits of BC 1 F 1 were normal and the grain hull was yellow (Figure 2D, d1).Segregation occurred amongst the BC 1 F 2 plants.Using grain hull color as an example, four plants (plant Nos. 6, 8, 12 and 19) produced black-hulled grains, but the remaining progeny had yellow-hulled grains.The grain-shattering trait also segregated into the normal and shattering phenotypes.The BC 1 F 2 progeny had differing levels of seed set after selfing; six individuals (25%) had less than 5% seed set whereas the remaining plants (75%) had a seed set greater than 5%.Plant Nos. 3 and 7 had very high seed sets (71.31% and 61.32%, respectively).

Meiotic Behavior of the BC 1 F 1 Plant
The BC 1 F 1 plant generally showed irregular meiosis.Univalent, bivalent, trivalent, quadrivalent and multivalent chromosomes could be observed (Figure 3, b-d).There were mainly four types of chromosome pairing at diakinesis/ metaphase I (Table 3).Lagging chromosomes were observed frequently in PMCs at metaphase I and anaphase I (Figure 3, e-f).Four lagging chromosomes were observed most frequently (42.31%) with one lagging chromosome seen the most infrequently (3.85%;Table 4).Chromosomal bridges were also observed in some PMCs at anaphase I (Figure 3, g).

GISH Analysis of the BC 1 F 1 Plant
The chromosome number in root tips of the BCF 1 plant was 2n = 48.GISH analysis indicated that four chromosomes were wholly labeled by the O. punctata probe.Two chromosomes were partly labeled (Figure 4, a1-2).In some diakinesis PMCs four univalents were fully labeled and two bivalents were partly labeled but the attached fragments were unlabeled (Figure 4, b1-2).At metaphase I of some PMCs, two partly labeled bivalents appeared in the equatorial plate, whilst four lagging chromosomes were labeled.In addition, there was another unlabeled lagging chromosome (Figure 4, c1-2).

Utilization of Desirable Genes from Wild Rice Resources
The wild Oryza species are an important source of desirable genes for resistance to major pests and diseases and for tolerance to various abiotic stresses.As such they form an extremely valuable genetic resource (Flagel et al., 2008;Multani et al., 2003).Researchers have attempted to use these wild resources for many decades.During the 1970's several interspecific hybrids and amphiploids were produced in order to introgress novel genes from wild species into cultivated rice (Khush, 1977).Many disease and insect resistance genes introgressed from wild species have been used in rice breeding and several varieties carrying these genes have now been released (Jena and Khush, 1989;Amante-Bordeos et al., 1992;Multani et al., 1994).Some wild species genes (Xa 21, Bph 18) have also been used in marker assisted selection (Singh et al., 2001;Jena et al., 2006).The purpose of this study was to obtain new rice germplasm and to introgress genes from O. punctata on a tetraploid level.In this investigation, the F 1 plants were sterile.However, improvements in three agronomically important traits were observed in the BC 1 F 1 and BC 1 F 2 plants-increased seed setting rate, normal shattering trait and short plant height.New polyploid rice lines (2n = 48), monosomic alien addition lines or gene introgression lines with O. punctata chromosome fragments or genes will now be obtained from this backcross progeny.

Effects of allopolyploidy in Crop Breeding
Polyploidy is an evolutionary innovation in many plant and some animal species (Ni et al., 2009).Allopolyploid plants are hybrids that contain two copies of the genome from each parent (Comai, 2000).Many commercially important crops such as wheat, cotton and canola are allopolyploids (A.R. Leitch, & I. J. Leitch, 2008;Chen, 2007).In addition, some allopolyploid rice species already exist in the wild.Synthetic allopolyploids should further enrich existing rice resources.Moreover, they will be beneficial for both the protection of wild resources and the storage and use of wild germplasms (Sangiacomo & Sullivian 1994;Kazi et al., 1996;Ge & Li, 2007;Goncharov et al., 2007;Yao et al., 2012).From an evolutionary perspective, different genomic combinations and polyploidization reflects the general direction of crop evolution (Cai et al., 2001).Allopolyploids possess the evolutionary advantage of having double the genetic material for growth and adaptation and can overcome the reproductive barrier of wild and cultivated species (Wang et al., 2013).Hence, they may play an important role in the distant hybridization and assist plant breeders in the utilization of genetic resources from wild species.

Conclusions
Obtaining desirable genes from wild species is an important approach in plant breeding.In combination with wild cross, polyploidization and backcrossing, the polyploids or allopolyploids will assist plant breeders in the utilization of genetic resources from wild species.

Figure 1 .
Figure 1.Scheme for production of backcross

Table 4 .
Lagging chromosomes of the meiotic metaphase I and anaphase I of the backcross-1 plant