Reproductive barriers in cassava: Factors and implications for genetic improvement

Abstract

Cassava breeding is hampered by high flower abortion rates that prevent efficient recombination among promising clones. To better understand the factors causing flower abortion and propose strategies to overcome them, we 1) analyzed the reproductive barriers to intraspecific crossing, 2) evaluated pollen-pistil interactions to maximize hand pollination efficiency, and 3) identified the population structure of elite parental clones. From 2016 to 2018, the abortion and fertilization rates of 5,748 hand crossings involving 91 parents and 157 progenies were estimated. We used 16,300 single nucleotide polymorphism markers to study the parents' population structure via discriminant analysis of principal components, and three clusters were identified. To test for male and female effects, we used a mixed model in which the environment (month and year) was fixed, while female and male (nested to female) were random effects. Regardless of the population structure, significant parental effects were identified for abortion and fertilization rates, suggesting the existence of reproductive barriers among certain cassava clones. Matching ability between cassava parents was significant for pollen grains that adhered to the stigma surface, germinated pollen grains, and the number of fertilized ovules. Non-additive genetic effects were important to the inheritance of these traits. Pollen viability and pollen-pistil interactions in cross- and self-pollination were also investigated to characterize pollen-stigma compatibility. Various events related to pollen tube growth dynamics indicated fertilization abnormalities. These abnormalities included the reticulated deposition of callose in the pollen tube, pollen tube growth cessation in a specific region of the stylet, and low pollen grain germination rate. Generally, pollen viability and stigma receptivity varied depending on the clone and flowering stage and were lost during flowering. This study provides novel insights into cassava reproduction that can assist in practical crossing and maximize the recombination of contrasting clones.

Fig 1. Cubic regression of weather variables (rainfall and minimum, average, and maximum temperatures) for the seed setting rate (seeds produced every hundred crossings) and maximum temperature for abortion rate.

The blue line is the cubic regression line, while the gray area refers to the confidence interval of the regression.

Fig 2. Average weather data for Cruz das Almas, Bahia, Brazil between 2010 and 2019 and the LOESS regression for average rainfall and temperature for seed setting.

(A) Effect of average temperature on seed setting: light green–maximum and minimum daily temperatures; green–average maximum and minimum daily temperatures; dark green–confidence interval for average daily temperature. (B) Effect of accumulated rainfall per 10-day intervals (mm) on seed setting.

Fig 3. Pollen viability of 16 cassava genotypes evaluated with 2% Alexander’s solution.

(A) Average and standard deviation of pollen viability of the different genotypes; (B) high pollen viability in the Aipim Abacate genotype; (C) low pollen viability in clone 7909–04, with viable (violet) and non-viable (green—arrow) pollen grains; (D) trimorphism of pollen grains in the BRS Novo Horizonte genotype. Bars: 1 mm.

Fig 4. Overview of cassava pistils stained with aniline blue and visualized by fluorescence microscopy 48 hours after pollination.

(A) Absence of germination and polymorphism in pollen grains from the BGM-0661 × BRS Jari cross; B) few pollen grains germinated on the surface of the stigmatic papillae with penetration of the pollen tube in the BGM-0661 × BGM-2167 cross; (C–D) pollen grains germinated on the surface of the stigmatic papillae with penetration of the pollen tube in the Aipim Abacate x BRS Novo Horizonte and BGM-0470 × BRS Novo Horizonte crosses, respectively; (E–F) presence of pollenkitt in the germinated pollen grains; (G) pollen tube growth ceased at the first third of the stylus in the 7909–04 × BGM-0661 cross; (H–I) pollen tubes within the stylet, showing the irregular deposition of callose plates; J) pollen tubes in the ovary with growth toward the ovary; (K–L) unfertilized ovule; (M–P) ovule being fertilized in compatible crosses (BGM-0728 × Aipim Abacate, BRS Novo Horizonte × Aipim Abacate, BGM1760 × BGM-2338, and BGM-2338 × BGM-0019, respectively). Bars: A–D, G–H, J–K, and M–P = 1 mm; E, F, I, and L = 200 μm.

Fig 5. Population structure and genomic relationship obtained via the analysis of 16,300 single-nucleotide polymorphisms among 86 cassava parents.

(A) Heat map of the genomic relationship matrix with the genotypes ordered according to the discriminant analysis of principal components (DAPC). The bar to the left of the heat map represents the DAPC group (i.e., Groups 1, 2 and 3 in red, blue and green, respectively). (B) The DAPC, in which the two axes of the upper graph represent the first two linear discriminants (LD). Each point represents an individual. The different subpopulations identified by the DAPC analysis are represented by the colors red, blue and green, while the bottom graph represents the distributions of the groups based on the first discriminant function only.

Fig 6. Correlation between the genomic relationship calculated via the analysis of 16,300 single-nucleotide polymorphisms versus abortion rate and seed setting rate in cassava.

The 86 cassava parents were previously grouped into three clusters based on the discriminant analysis of principal components (DAPC), and crosses were performed between parents of the same cluster (i.e., 1x1, 2x2 and 3x3) or between clusters (i.e., 1x2,1x3 and 2x3).