Unexpected Inheritance Pattern of Erianthus arundinaceus Chromosomes in the Intergeneric Progeny between Saccharum spp. and Erianthus arundinaceus

Erianthus arundinaceus is a valuable source of agronomic traits for sugarcane improvement such as ratoonability, biomass, vigor, tolerance to drought and water logging, as well as resistance to pests and disease. To investigate the introgression of the E. arundinaceus genome into sugarcane, five intergeneric F1 hybrids between S. officinarum and E. arundinaceus and 13 of their BC1 progeny were studied using the genomic in situ hybridization (GISH) technique. In doing so, we assessed the chromosome composition and chromosome transmission in these plants. All F1 hybrids were aneuploidy, containing either 28 or 29 E. arundinaceus chromosomes. The number of E. arundinaceus chromosomes in nine of the BC1 progeny was less than or equal to 29. Unexpectedly, the number of E. arundinaceus chromosomes in the other four BC1 progeny was above 29, which was more than in their F1 female parents. This is the first cytogenetic evidence for an unexpected inheritance pattern of E. arundinaceus chromosomes in sugarcane. We pointed to several mechanisms that may be involved in generating more than 2n gametes in the BC1 progeny. Furthermore, the implication of these results for sugarcane breeding programs was discussed.


Introduction
Sugarcane (Saccharum spp.) plays a pivotal role in world agriculture as a primary sugar-producing crop and has significant potential as a renewable bioenergy crop [1]. The genus Saccharum is comprised of six species: The two wild species are S. spontaneum and S. robustum, and the four cultivated species are S. officinarum, S. barberi, S. sinense and S. edule. S. officinarum (2n = 80) is known as the noble cane due to its high sugar content and thick and juicy culms. The wild species S. spontaneum (2n = 40-128; chromosome number varies) has very low sugar content but exhibits high vigor, profuse tillers and strong ratooning ability, as well as resistance to diseases and pests. Modern sugarcane cultivars are highly complex polyploid aneuploids and typically have 100-130 chromosomes derived from a combination of these two species [2].
During the early 20 th century, interspecific hybridization was used to introgress desirable traits from wild species into sugarcane cultivars, and this practice led to substantial improvements in sugarcane agriculture [3]. In particular, interspecific hybridization between S. officinarum as the female parent and S. spontaneum as the male parent, followed by successive backcrosses of the hybrids to different clones of S. officinarum as the recurrent parent, significantly increased cane yields and resistance to biotic and abiotic stresses. In this hybridization strategy, F 1 hybrids and plants in the first backcross generation (BC 1 ) receive 2n gametes from female parent and n gametes from male parent, and plants in the second backcross generation (BC 2 ) receives n gametes from both the female and male parents [4]. The purpose of the process termed nobilization, which refers to the crossing and backcrossing of intergenic hybrids to noble cane, was to retain high-sugar producing clones and to eliminate the negative effects of wild germplasm [5]. Over the past decades, much insight has been gained into the mechanisms underlying 2n gamete formation through the use of molecular genetic and cytological techniques. Using simple sequence repeat (SSR), amplified fragment length polymorphism (AFLP) and diversity arrays technology (DarT), Hermann et al. [6] provided molecular marker data that suggested the mechanism was second division restitution (SDR) or megaspore tetrad cell fusion (MTCF). Bielig et al. [7] provided cytological evidence that 2n male gamete formation was probably attributable to SDR. Nevertheless, the mechanisms underlying 2n gamete formation in sugarcane are still not fully understood.
Indeed, the occurrence of 2n gametes is not rare in the plant kingdom and it has been reported in many genera, including Brassica, Paspalum, Brachiaria, Citrus, Fragaria, Malus, Manihot, Medicago, Solanum, and Trifolium [8][9][10][11]. Several explanations for 2n gamete formation have been proposed, including premeiotic and post-meiotic genome doubling, and meiotic restitution. Among these possibilities, the majority of reports have identified a restitution of the meiotic cell cycle in several species [12][13][14], suggesting that it is the predominant mechanism of 2n gamete formation in plants. However, a small number of reports have documented pre-meiotic and post-meiotic genome duplications, indicating that these mechanisms of 2n gamete formation are quite rare. Three cytological processes can lead to 2n gamete formation during abnormal meiosis: first division restitution (FDR), second division restitution (SDR) and indeterminate meiotic restitution (IMR). In FDR, homologous chromosomes remain together when the nucleus fails to divide after telophase I, and after a normal second division, sister chromatids derived from each chromosome move to opposite poles. In SDR, normal separation of the homologous chromosomes at first division is followed by the absence of the second meiotic division and sister chromatids fail to migrate to opposite poles at second division. IMR has been best described in lily meiocytes, and simultaneously shows characteristics similar to both SDR and FDR within a single meiocyte [15][16][17][18].
Modern sugarcane cultivars have limited genetic diversity, due to the small number of progenitors used in the initial interspecific hybridizations during the process of nobilization [19][20][21]. This genetic bottleneck has impeded further sugarcane improvement for certain traits such as tolerance to biotic and abiotic stresses. Therefore, it is urgent to broaden the genetic base of sugarcane by introgressing favorable genes from closely related Erianthus, Miscanthus, Narenga and Sclerostachya genera [22].
Erianthus is one of the most closely related genera to Saccharum and has attracted the interest of sugarcane breeders worldwide. Erianthus arundinaceus (E. arundinaceus, 2n = 20, 40, 60) is one of eight species in the genus Erianthus [23], and it possesses valuable agronomic traits for sugarcane improvement such as high biomass, vigor, ratoonability, tolerance to drought and water logging, and resistance to pests and disease [23][24][25][26][27][28]. Favorable alleles can be introduced into modern sugarcane cultivars for yield and stability improvement, although the hybrid progeny is often sterile [26]. However, significant progress has been made to produce genuine F 1 hybrids and to backcross the progeny successfully. Molecular markers and genomic in situ hybridization (GISH) techniques have been used to identify true intergeneric hybrids between Saccharum spp. and Erianthus spp. [23,24,26,29]. According to histological staining character of root tips, Fukuhara et al. [30] concluded that F 1 hybrids were successfully obtained from the intergeneric hybridization between Saccharum spp. hybrid and E. arundinaceus.
Using GISH, N. Piperidis et al. [29] reported that chromosome transmission was n+n in both F 1 (S. officinarum 6 E. arundinaceus) and BC 2 (BC 1 6 sugarcane cultivar) generations, but was 2n+n in the BC 1 (F 1 6sugarcane cultivar) cross. In similar crosses six F 1 hybrids had fewer than 70 chromosomes and one had more than 70, indicating that all F 1 crosses were aneuploidy [26]. In this report, we studied chromosome transmission in (E. arundinaceus 6 S. officinarum) hybrids by using GISH to determine the chromosome composition of two generations including five intergeneric F 1 hybrids and 13 BC 1 progeny.

Plant materials
The plant materials used in this study consisted of 18 clones derived from two generations of intergeneric hybrids ( Table 1). The male parent of the F 1 generation was either E. arundinaceus

Aneuploidy in F 1 hybrids
Five F 1 hybrids analyzed by GISH were characterized by the presence of 68-69 chromosomes, consisting of 40 Saccharumderived chromosomes and 28-29 E. arundinaceus-derived chromosomes ( Figure 1A, Figure S1-S4 in File S1). Therefore, the five F 1 hybrids were the products of n+n chromosome transmission (Table 2), and all hybrids were also aneuploid. These results are consistent with G. Piperidis's studies [26]. Notably, the high rate of aneuploidy in F 1 hybrids contributes to the production of unbalanced gametes, which might be associated with the high degree of sterility in F 1 hybrids [31,32]. Chromosomes inherited from divergent parents are often unable to pair with each other in meiosis [33][34][35][36], producing very few or no viable pollen grains [26,37]. In order to obtain backcross generations, an adjustment in sugarcane breeding was implemented, involving F 1 hybrids as female parents and CP 84-1198 as the male parent, although this does not conform perfectly to the fundamental principles of backcross breeding. Even though F 1 hybrids were used as female parents, their fertility was still very low. Among the five F 1  Unexpected inheritance pattern in BC 1 progeny GISH analysis of nine BC 1 progeny revealed plants with a total chromosome complement ranging from 117 to 125 (Table 2), of which 93 to 97 chromosomes were derived from Saccharum and 22 to 29 chromosomes were derived from E. arundinaceus ( Figure 1B, Figure S5-S12 in File S1). These results indicated that the nine BC 1 progeny were products of 2n+n transmission. G. Piperidis et al. [26] and N. Piperidis et al. [29] reported similar results. GISH analysis of another four BC 1 progeny revealed plants with a total chromosome complement ranging from 116 to 132, and evidence of an unusual mode of chromosome transmission (Table 2, Figure 1C-1F). In YCE 01-36, YCE 01-61, YCE 01-69 and YCE 01-92, 85 to 96 chromosomes were derived from Saccharum and 36, 31, 31 and 35 chromosomes were derived from E. arundinaceus, respectively. These results indicated that, in these four BC 1 progeny, more than 29 E. arundinaceus-derived chromosomes (Table 2, Figure 1) were transmitted, which is a greater number than was detected in the F 1 generation. To our knowledge, ours is the first report to document that the E. arundinaceus-derived chromosome number was above 29 in BC 1 progeny. Within the plant kingdom, this unusual phenomenon has rarely been reported. It is especially noteworthy that four BC 1 progeny out of 13 exhibited greater than 2n female-inherited chromosomes, suggesting that this newly discovered phenomenon can occur at relatively high frequency in sugarcane (above 30%). More importantly, the occurrence of more than 2n female gametes was detected in two diverse parental combinations rather than an individual case, suggesting that this phenomenon is not restricted to an individual plant. The results also suggest that the four BC 1 progeny were the product of a new pattern of chromosome transmission. Interestingly, YCE 01-36 ( Figure 1C) and YCE 01-92 ( Figure 1F) were found to both have a terminally translocated chromosomes.

Possible mechanisms
The unexpected inheritance pattern that we observed in BC 1 progeny is not in accordance with prevailing theories of chromosome transmission in hybrids. If meiosis occurs normally, four gametes are generated with different numbers of E. arundinaceus-derived chromosomes, due to the 29 E. arundinaceus chromosomes in F 1 hybrids. As a result, two gametes are produced containing 14 E. arundinaceus chromosomes and the other two gametes contain 15 E. arundinaceus chromosomes. (Figure 2A). In FDR, two gametes are produced with 29 E. arundinaceus chromosomes ( Figure 2B). In SDR, one gamete is produced with 28 E. arundinaceus chromosomes and a second contains 30 E. arundinaceus chromosomes ( Figure 2C). SDR has been extensively reported in sugarcane [7,38,39]. In addition, Narayanaswami [40] discovered that 2n gametes originated from the fusion of the two innermost cells of the megaspore tetrad (megaspore tetrad cell fusion, MTCF). Post-meiotic restitution (PMR), in which chromosome doubling occurs after the second meiotic division, was observed by Bremer [38].
The normal separation of chromosomes in the first meiotic division or sister chromatids in the second meiotic division is called disjunction. Nondisjunction can occur in the first meiotic division (nondisjunction of homologous chromosomes; NHC) or second meiotic division (nondisjunction of sister chromatids; NSC). These distinct processes of nondisjunction create gametes with different numbers of chromosomes ( Figure 2D, Figure 2E). In this study, we propose two possible mechanisms responsible for the formation of gametes with chromosome number greater than 2n. The first possibility (Model I) involves both NHC and SDR, which would generate two gametes with different even numbers of E. arundinaceus chromosomes after meiosis ( Figure 2F; NHC + SDR). The second possibility (Model II) involves both FDR and NSC, which would generate two gametes with different odd or even numbers of E. arundinaceus chromosomes after meiosis ( Figure 2G; FDR + NSC).
According to the results obtained from plants in the F 1 generation, their meiocytes contained 29 E. arundinaceus chromosomes. During S phase of pre-meiotic interphase, all chromosomes are duplicated and each chromosome is comprised of two sister chromatids. Consequently, after meiosis, the total number of E. arundinaceus chromosomes in the F 1 gametes should be 58. According to Model I, if there are i pair(s) of NHC in the first meiotic division, this yields a difference of 2(2i +1) E. arundinaceus chromosomes between the two gametes. According to Model II, if there are j chromosomes with NSC in the second meiotic division, this yields a difference of 2j E. arundinaceus chromosomes between the two gametes. Thus, the following two simultaneous linear equations are obtained: Model I : In these equations, g and g' are the total number of E. arundinaceus chromosomes in each gamete after meiosis, and i and j are the number of NHC and NSC, respectively. From our experimental observations of 29 E. arundinaceus chromosomes in a meiocyte and 58 E. arundinaceus chromosomes in two gametes, we required that 0 # g # 58; 0 # g' # 58; 0 # i # 14; and 0 # j # 29. The four variables g, g', i and j were integral. After solving these simultaneous linear equations, we obtained two formulas: Model II : g~2jz29 Based on these formulas, graphs of these linear equations are shown in Figure 3 and Figure 4.
In this study, eight different E. arundinaceus chromosome numbers were observed (22, 23, 27, 28, 29, 31, 35 and 36) in BC 1 progeny. Due to the fact that we detected odd numbers of E. arundinaceus chromosomes in BC 1 progeny, we speculate that Model II may be a more likely mechanism than Model I, but Model I cannot be ruled out as a mechanism occurring in plants that inherited even numbers of E. arundinaceus chromosomes. It is possible that both models are valid, suggesting that both mechanisms can occur.
Modern sugarcane cultivars are characterized by a high degree of inbreeding depression, so any increase in the heterozygosity of gametes may be beneficial to breeding efforts [6]. Depending on the specific mode of chromosome segregation, gametes can exhibit different degree of heterozygosity. During normal meiosis, crossing-over occurs between two non-sister chromatids. In FDR, 2n gametes always possess two non-sister chromatids and consequently maintain equivalent levels of parental heterozygosity and epistatic interactions. In SDR, sister chromatids do not separate and these gametes exhibit high levels of homozygosity. As a result, most parental heterozygosity and epistatic interactions are lost [41,42]. The highest degree of heterogeneity is found in gametes originated through IMR, since these gametes result from a mixture of FDR and SDR [15]. In addition, when chromatids migrate to the same pole as in NHC and NSC, chromosomes are doubled. In NHC+SDR or FDR+NSC, some E. arundinaceus chromosomes would be doubled twice. This process likely creates a larger number of new multilocus allelic combinations and provides the opportunity to select the resulting germplasm for new, desirable traits.

Future directions
In order to understand the underlying mechanisms involved in generating the number of E. arundinaceus chromosomes in BC 1 progeny, detailed cytological observations of female gametes and chromosomal dynamics in the embryo sac of F 1 hybrids are needed. Although difficult to access, a more thorough understanding of the megagametophyte may result in possible applications for improving sugarcane through 2n gamete transmission.