Rice (Oryza sativa
) is a staple food in West Africa, where its demand keeps increasing due to population growth. Hence, there is an urgent need to identify high-yield rice cultivars that fulfill this demand locally [1
]. Hybrid rice can contribute to food security by increasing the availability of staple food for consumption by farm households and increasing on-farm income through higher grain yield [2
]. Success in breeding and growing hybrid rice commercially in China has caused great interest among rice breeders throughout the world. However, the natural outcrossing rate in rice cultivars is extremely low and hybrid seed production, using male sterility or gametocides, presents some difficulties. Increasing the potential outcrossing rate would facilitate the use of hybrids in other areas of the world.
The availability of both stable cytoplasmic male sterility (CMS) and fertile restoration system is very important for commercial exploitation of heterosis in any crop. The advantage of cytoplasmic male sterility (CMS) is that the pollen in the male sterile line is aborted, while the female organ is normal. This allows stigma cross-pollination from other plants to generate heterozygous seeds [3
]. Stigma exertion, panicle exertion, and angle of opened lemma and palea of male sterile lines are the key determinants of outcrossing in hybrid rice seed production [4
]. The exerted stigma can bypass the physical barrier of glumes between the pollen of restorer lines and the stigma of CMS lines in rice. Exerted stigma of rice seems to improve the outcrossing rate of the sterile lines [6
]. Agronomic traits should also be taken into account for achieving heterosis for grain yield. Hence, developing CMS lines with good floral and agronomic traits is necessary for hybrid rice breeding.
The floret of O. sativa
is a terminal panicle of perfect flowers. Each spikelet has a branched stigma, six stamens, and two lodicules. At blooming, the flowers open rapidly. The flowers remain open for 1–3 h and close after anthesis, but never reopen. Some floral characteristics, such as stigma size, stigma exertion, and flower opening duration, affect the rate of outcrossing in rice. Usually the stigma remains inside the glumes during and after anthesis. In some cultivars, some stigmas remain outside the glumes after anthesis and fertilization. The rate of exerted stigma, which ranges from 0 to 90%, is correlated with stigma length. Stigma length ranges between 0.4 and 1.6 mm [8
]. The duration of receptivity is variable and may exceed five days.
Achieving improved floral and agronomic traits in the maintainer line is the first step in developing new CMS lines with desirable traits through the backcross breeding method. Evaluation of newly developed CMS lines of rice for their morphological and floral traits to determine variation among genotypes is essential so that phenotypically excellent lines can be exploited for the development of rice hybrids to identify genotypes with desirable morphological and floral characteristics [5
]. The genetics of stigma exertion is quantitative and influenced by dominance effects, followed by additive effects [13
]. The genetic advance for stigma length and spikelet length appears to be greater than that of other floral characteristics, thus stigma exertion could be improved by selection in early generations.
In 2010, AfricaRice began breeding hybrid rice using both cytoplasmic male sterile and environmental genetic male sterile systems [14
]. In this study, 216 maintainer lines were evaluated for their floral and agronomic characteristics. Five successful backcrosses with 15 selected maintainer lines led to developing 15 new CMS lines. The 15 new CMS were evaluated for outcrossing rates and agronomic characteristics along with popular CMS lines as checks. The main objective of this research was to study the genetic variability of some floral and agronomic traits of newly bred CMS lines in rice.
2. Materials and Methods
This research was carried out at AfricaRice Regional Center, Saint Louis, Senegal from 2013 to 2017, as described briefly in Figure 1
. The study included 216 maintainer lines (F6
) derived from four breeding populations, namely V20B/IR72793B (46 lines), IR80561B/IR72793B (54 lines), IR93561B/IR58025B (65), and IR93561B/IR68897B (51 lines), which were evaluated for agronomic and floral characteristics using an augmented design with 12 blocks. Each block contains 18 lines and two popular maintainers IR68897B (early maturity) and IR58028B (late to medium maturity) as checks. Each line was transplanted in four rows 3 m long and with a spacing of 20 × 20 cm. The experiment used a recommended package of crop husbandry practices during plant growth. Data were recorded on five random plants within each plot for number of days to 50% flowering (DAF), number of days to maturity (DAM), stigma exertion (SE, %), panicle length (PnL), panicle weight (PnW), spikelet fertility (SF, %), panicle exertion (PnE, %), tillers per plant (NPT), plant height (PH) grains per panicle (NGP), grain yield per plant (GYP), and 1000-grain weight (GW).
2.1. Backcrossing and Derived Offspring Assessment
Out of 216 maintainer lines evaluated for floral and agronomic traits, 15 lines were selected and testcrossed with IR58025A (a popular WA CMS used for commercial production in many countries). Five consecutive backcrosses (BC1:5) were conducted to complete the transfer of cytoplasmic male sterile (CMS) to selected maintainer lines. The backcross offspring and its parent were transplanted side by side on two or three rows based on the seeds available. Pollen fertility (POF), DAF, and phenotypic acceptability (PAC) were evaluated for all offspring and their parents. The new CMS lines derived from BC5:6 were evaluated for agronomic and floral characteristics.
2.2. Outcrossing Experiment
The 15 newly-bred CMS lines derived from BC5:6
, plus two old CMS lines and their respective maintainers, were evaluated for outcrossing rates and agronomic characteristics during the dry and wet seasons in 2017. The maintainer of each line was sown four days after the male sterile lines to optimize the synchronization of flowering. The CMS and their maintainer were evaluated in a randomized complete block design with three replications. The CMS lines were transplanted in six rows, while their respective maintainer used two rows of 3 m long; i.e., 6:2 row ratio. Flag leaf cutting was done for all CMS before heading to facilitate outcrossing. Correlation coefficients between floral characteristics and outcrossing rates were estimated using SPSS version 16 for Windows [16
Data were recorded on five plants per plot for DAF, DAM, SE, PnE (%), PnL, PnW, SF, NPT, PH, NGP, GYP, and GW. About 15 spikelets from the freshly emerged panicles of at least 10 plants were collected and examined under a microscope with 1% iodine potassium iodide (IKI) solution for pollen sterility assessment [17
]. For this purpose, panicles emerging from the sheath were bagged with paper bags prior to anthesis to prevent cross-pollination. Data recording was done according to the standard evaluation system for rice [18
]. For glume angle, at the time of flowering, three well-opened florets of the primary panicle were collected. The angle of the opened floret was measured using a protractor. The stigma exertion was calculated by the ratio of spikelets with exerted stigma to the total number of spikelets and expressed as a percentage. Bagged panicles were also harvested to assess spikelet percentage. Digital cameras were used to take photos every 4 min to estimate the flowering time of each CMS lines.
Estimation of phenotypic and genotypic variances:
The phenotypic and genotypic coefficients of variation were computed as per the methods suggested by [19
Genotypic variance (σ2g) = (Msg − Mse)/r
Environmental variance (σ2e ) = Mse
are the mean sum of squares for the genotypes and error in the analysis of variance, respectively, and r is the number of replications.
The phenotypic variance was estimated as the sum of the genotypic and environmental variances:
Phenotypic variance (σ2ph) = σ2g + σ2e
Estimation of genotypic and phenotypic coefficient of variability:
The genotypic and phenotypic coefficients of variability were calculated according to the following formulae:
Genotypic Coefficient of Variation (GCV) = (σg /grand mean) × 100
Genotypic Coefficient of Variation (PCV) = (σph /grand mean) × 100
are the genotypic and phenotypic standard deviations, respectively.
Heritability and genetic advance:
Heritability in a broad sense (h2b
) for all characters was computed as per the following formula:
The genetic advance for selection intensity (k) at 5% was estimated by the following formula [21
EGA = the expected genetic advance under selection;
σph = the phenotypic standard deviation;
h2b = heritability in a broad sense;
k = selection intensity.
Genetic advance per population mean = (EGA/grand mean) × 100
Low seed set remains a major challenge in hybrid seed production for rice. Improvement of floral characteristics, especially stigma and panicle exertion, of CMS lines is very important to improving outcrossing rates and increasing seed set for producing hybrid seed. This requires us to develop maintainer lines with desired floral and agronomic characteristics and transfer CMS through backcrossing. We were able to breed new CMS lines with outcrossing rates above 50%. Out of 216 maintainer lines evaluated for floral and agronomic characteristics, 16 were selected for further use in breeding.
There were highly significant differences among the 216 F6
maintainer lines for all characteristics except angle of floret opening (°) and number of tillers per plant, thus indicating that these lines differed significantly for most desired traits. Similar results were obtained by the authors of [23
], who found highly significant differences for various traits among 11 genotypes. Significant variation was observed for all characteristics, which provides a source for further breeding of maintainer lines combining the desired agronomic and floral characteristics. The large variation in days to 50% flowering and maturity of maintainers and CMS is very important to optimize the synchronization of flowering with restorer lines of different duration in hybrid seed production. The ratio of GCV to PCV was high, thus indicating that most of the phenotypic expression of the agronomic and floral characteristics was due to genetics. This finding suggests the potential for genetic gains due to selection for these traits in such CMS lines of rice. The results also indicated significant genetic variability among the tested maintainer lines for most characteristics, which agreed with previous research [24
]. Broad-sense heritability estimates were relatively high for all characteristics, thereby indicating that selection of superior genotypes based on phenotypic performance should be very effective. [26
]. The high heritability estimates along with high genetic advance should allow for selecting the best offspring among CMS lines, which also agreed with previous results elsewhere [27
]. Low heritability estimates along with low genetic advance reveal a non-additive type of gene action and significant genotype × environment interaction affecting the expression of characteristics [25
The newly-bred CMS lines exhibited highly significant differences for most characteristics. Most of them exhibited desirable traits vis-à-vis the two checks. The high outcrossing rate associated with high stigma and panicle exertion indicated that selection for such floral characteristics along with spikelet angle opening could be efficient for improving outcrossing rates. These results agreed with previous research [12
], in which natural outcrossing rates ranged from 14.8 to 51%, while 14 of our newly developed CMS lines had an outcrossing rate above 35%. Our results suggest that additive genes play a major effect on floral and agronomic characteristics, thus being selected in earlier generations was very effective, as noted previously by Tian et al. [29
Highly significant positive correlations were observed between different traits with outcrossing rates, as also found elsewhere [31
]. This finding suggests that indirect selection to increase outcrossing rates may be effective in our breeding population for hybrid rice. A high panicle exertion in CMS lines was essential to attain high outcrossing rates, as noted previously [34
]. Plant height, panicles per plant, and panicle length should also be regarded as very important for enhancing outcrossing rates in rice [35