Analysis of inbreeding depression in five S<sub>1</sub> Cassava families of elite varieties at the seedling nursery and clonal evaluation trial stages.

Lydia Jiwuba; Alex Ogbonna; Ugochukwu Ikeogu; Favour Okeakpu; Kenneth Eluwa; Eunice Ekaette; Damian Njoku; Peter Okocha; Chiedozie Egesi; Emmanuel Okogbenin

doi:10.12688/f1000research.153922.1

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Research Article

Analysis of inbreeding depression in five S₁ Cassava families of elite varieties at the seedling nursery and clonal evaluation trial stages.

[version 1; peer review: 1 approved, 1 approved with reservations]

Lydia Jiwuba¹, Alex Ogbonna², Ugochukwu Ikeogu², [...] Favour Okeakpu¹, Kenneth Eluwa¹, Eunice Ekaette³, Damian Njoku¹, Peter Okocha⁴, Chiedozie Egesi^1,2,5, Emmanuel Okogbenin⁶

Lydia Jiwuba¹, Alex Ogbonna², [...] Ugochukwu Ikeogu², Favour Okeakpu¹, Kenneth Eluwa¹, Eunice Ekaette³, Damian Njoku¹, Peter Okocha⁴, Chiedozie Egesi^1,2,5, Emmanuel Okogbenin⁶

PUBLISHED 27 Aug 2024

Author details Author details

¹ Biotechnology, NRCRI, National Root Crops Research Institute, Umudike, 7006, Nigeria
² Department of Plant breeding and genetics, Cornell University, Ithaca, New York, 14850, USA
³ NABDA, National Biotechnology Development Agency, Airport Road, Abuja, Nigeria
⁴ College of Crop and Soil Science, Michael Okpara Univeristy of Agriculture, Umudike, Nigeria
⁵ Biosciences, International Institute of Tropical Agriculture, Ibadan, Oyo, Nigeria
⁶ AATF, African Agricultural Technology Foundation, Nairobi, Kenya

Lydia Jiwuba
Roles: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Resources, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

Alex Ogbonna
Roles: Data Curation, Formal Analysis, Writing – Review & Editing

Ugochukwu Ikeogu
Roles: Formal Analysis, Methodology, Resources, Writing – Review & Editing

Favour Okeakpu
Roles: Methodology, Resources, Writing – Review & Editing

Kenneth Eluwa
Roles: Methodology, Resources

Eunice Ekaette
Roles: Methodology, Resources, Writing – Review & Editing

Damian Njoku
Roles: Methodology, Writing – Review & Editing

Peter Okocha
Roles: Supervision, Writing – Review & Editing

Chiedozie Egesi
Roles: Investigation, Resources, Supervision, Visualization, Writing – Review & Editing

Emmanuel Okogbenin
Roles: Conceptualization, Funding Acquisition, Investigation, Methodology, Project Administration, Resources, Supervision, Validation, Visualization, Writing – Review & Editing

OPEN PEER REVIEW

REVIEWER STATUS

This article is included in the Plant Science gateway.

Abstract

Background

Cassava is an outcrossing, highly heterozygous plant that is reported to suffer from inbreeding depression. However, unraveling recessive traits and exploring additive genes would require a limited level of inbreeding for the genetic improvement of cassava.

Method

The impact of inbreeding depression (ID) on agronomic and biotic tolerance of cassava was evaluated using the S1 progenies of five African cassava varieties (TMS 30572, TME 419, TMS 98/0505, TMS 01/1371, and TMS 98/0002) at the seedling and clonal evaluation stages.

Results

At both trial stages, the effects of ID were severe on average performance of fresh root yield and fresh foliage yield; moderate on harvest index, dry matter content, vigor, and tolerance to cassava mosaic disease, cassava bacterial blight, and cassava anthracnose diseases; and less severe on plant height. Further examination of S1 families using molecular markers showed varying levels of heterozygosity at several loci across the genome. In addition, the degree of heterozygosity and ID varied by the S1 family. The TMS 01/1371 family showed the lowest degree of heterozygosity and ID on the average performance of different agronomic attributes, indicating that inbreeding may be strategically explored in this family to increase genetic gain and identify useful recessive traits. Lastly, the observed depression from inbreeding was higher in seedling evaluation than in clonal evaluation trials for fresh root yield, fresh foliage yield, harvest index, and dry matter content.

Conclusion

S1 individuals showing relatively low heterozygosity based on SNP data were selected as parents to advance genetic gain in cassava. Generally, reduced heterozygosity was prominent in traits with severe ID impacts on average phenotypic performance. Our results highlight the relative importance of exploring non-additive genetic effects and transgressive segregations for favorable allele combinations in cassava improvement.

Keywords

Cassava, Seedling nursery stage, Clonal evaluation trial, Inbreeding depression, S1 progenies, Heterozygosity index, SNPs markers, Cassava improvement

Corresponding author: Emmanuel Okogbenin

Competing interests: No competing interests were disclosed.

Grant information: This study was conducted with funding support from the Generation Challenge Program (GCP) (grant number G7010.01.05) and the National Root Crops Research Institute (NRCRI), Umudike, Nigeria .
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2024 Jiwuba L et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

How to cite: Jiwuba L, Ogbonna A, Ikeogu U et al. Analysis of inbreeding depression in five S₁ Cassava families of elite varieties at the seedling nursery and clonal evaluation trial stages. [version 1; peer review: 1 approved, 1 approved with reservations]. F1000Research 2024, 13:966 (https://doi.org/10.12688/f1000research.153922.1) First published: 27 Aug 2024, 13:966 (https://doi.org/10.12688/f1000research.153922.1) Latest published: 27 Aug 2024, 13:966 (https://doi.org/10.12688/f1000research.153922.1)

Introduction

Cassava (Manihot esculenta Crantz) plays a vital role as one of the primary sources of energy in the diets of most tropical countries. Additionally, its significance has grown, as it now serves as a crucial raw material for diverse industries. Consequently, understanding its genetics and factors influencing trait inheritance and expression is of utmost importance for its continued development.

Inbreeding occurs when two genetically related individuals mate, increasing the likelihood of their offspring having recessive or deleterious traits. This often results in a reduced fitness of a population, known as inbreeding depression. Cassava is highly heterozygous and typical cross-pollinated species, has been associated with severe inbreeding depression (Rojas et al., 2009). Nonetheless, it is encouraging to observe that interest in inbreeding is beginning to gain momentum (Ceballos et al., 2004, 2007; Rojas et al., 2009) in cassava improvement initiatives. Certainly, there is the potential to harness the benefits of inbreeding, similar to what has been achieved with maize, by capitalizing on both additive and non-additive effects (Rojas et al., 2009). Inbreeding will provide several advantages in cassava such as elimination of harmful recessive genes. Through selection, the less desirable genes are eliminated and superior genes are begin accumulated resulting in an increase in the productivity of the inbred populations (Ceballos et al., 2004). A key objective of this study was to explore the means of efficiently utilizing additive genetic variance in trait development through the fixation of beneficial alleles for key economic traits. By fixing useful alleles, we aimed to create valuable genetic stocks that can serve as parent lines, thereby reducing segregation issues that typically arise when evaluating F₁ populations in breeding schemes for the development of improved varieties in Africa. This report presents the findings of our investigations for quantifying inbreeding depression in commercial elite African cassava lines. With these insights, we hope to pave the way for leveraging inbreeding as a strategic tool to enhance cassava breeding programs and to unlock its full potential as a vital crop for sustainable development.

Method

Study site

The field experiment was conducted at the National Root Crops Research Institute, (NRCRI) Umudike, Nigeria, while genotyping was conducted at the biotechnology laboratory of KBiosciences, Hoddesdon, London, United Kingdom. The NRCRI, Umudike, is located at latitude 5°30’N and longitude 7°30’E. It is at an altitude of approximately 122m above sea level and has an annual rainfall of approximately 2,166.3 mm, a temperature range of 22°C - 32°C, and a relative humidity range of 80–90%.

Population development

• Seed production
Five of the elite cassava genotypes (TMS 30572, TME 419, TMS 98/0505, TMS 01/1371 and TMS 98/0002) were obtained from the germplasm of International Institute of Tropical Agriculture, (IITA) Ibadan. These five elite cassava genotypes were used as progenitors (S₀) to generate S₁ progenies. They were not randomly selected because they were preferred for their wide adoption by farmers for favorable morphological and agronomic attributes in the elite germplasm. The elite genotypes were planted at a distance of 1 m × 1 m using 20 stem cuttings per genotype. For only self-pollination to occur, the plots were isolated by 100m apart from the neighbouring cassava plots. Self-pollination was performed for each elite clone. The pollinated flowers were secured within pollination bags in order to safeguard them from external pollen carried by bees. The bags were then carefully removed the following day. The mature fruits were harvested at 90 days after pollination, placed in a well-labelled pollination bags, and are allowed to dehiscent.
• Establishment of the seedling evaluation trial
Harvested S₁ botanical seeds were allowed a two - month dormancy period before being sowed in nurseries in a screen house. The seeds were sown in trays filled with sterilized soil with a mixture of loamy and sandy soil at a ratio of 2:1 in the screen house. Seeds germinated quickly under optimal soil temperatures (30°C–35°C) and moisture regimes. They were irrigated twice daily, in the morning and evening. The seeds started germinating 10 to 12 days after planting and were transplanted when they attained a height of 15–20 cm. After two months in the nursery, S₁ seedlings were transplanted to a well-prepared field where they were grown and evaluated. Harvesting was performed 12 months after planting, after which they were cloned to generate at least 10 stem cuttings per seedling for clonal evaluation.
• Establishment of the clonal evaluation trial
In the clonal evaluation trial, ten cuttings of each S₁ genotype were planted in the field for evaluation. All progenies belonging to the same family, together with the parents, were established in the same block in an augmented (Federer, 1956) experimental design. In this design, each clone or genotype was planted in a row and are not replicated while the parents serve as checks and are replicated in each block. The spacing within rows was 1 m, and the row spacing was 1.5 m to minimize inter-plot interference.

Single nucleotide polymorphisms (SNPs) markers genotyping

Genomic DNA was extracted from the leaf samples of each individual of the S₁ population (from five families) and from the parents, as described by Dellarporta et al. (1983). A total of 200 SNPs markers with good genome distribution and coverage were analyzed in 260 DNA samples. Constituent reagent volumes for the KBioscience Competitive Allele-specific PCR genotyping system (KASP) genotyping mix were as follows:

• 5 μl DNA
• 5 μl KASP 2X reaction mix
• 12 μl of Allele-specific primer 1 (100 μM)
• 12 μl of Allele-specific primer 2 (100 μM)
• 30 μl of common (reverse) Primer (100 μM)
• 46 μl of H₂O/Tris-HCl (10 mM, pH 8.3)

Procedure

5 μl DNA samples were arrayed on a PCR plate. The DNA was genotyped as a liquid sample. The KASP genotyping mix was dispensed robotically into PCR plates containing 5 μl DNA samples and briefly vortexed. The plate was sealed with an optically clear seal and thermally cycled using a Peltier block-based thermocycler at 94°C for 15 minutes. The plate was read using a suitable fluorescent plate reader at ambient temperature. Genotyping data were analyzed using the most FRET-capable plate readers.

Data collections

• Pests and diseases measurement

During the growth period of the seedling nursery and clonal trials, the plants were evaluated for Cassava Mosaic Disease (CMD), cassava bacterial light (CBB), Cassava Anthracnose Disease (CAD), and vigor 1, 3, 6, 9, and 12 Months After Planting (MAP).

A. Vigor was evaluated based on a scale of 1-5; where 1 = very weak (plants somewhat stunted with very thin stems), 2 = weak (thin stem), 3 = intermediate (moderately growing), 4 = vigorous (fast growing and no bending), and 5 = extra vigorous (very fast growing, strong, and no bending) (IITA, 1990).
B. Cassava Mosaic Disease (CMD) was visually scored on a scale of 1-5; where 1 = no symptoms observed; 2 = mild chlorotic pattern on the entire leaflet; 3 = strong mosaic pattern on the entire leaf; 4 = severe mosaic/distortion of 2/3 of leaflet; and 5 = severe mosaic/distortion of 4/5 of leaflet.
C. Cassava Bacterial Blight (CBB) was visually scored on a scale of 1-5; where 1 = no symptoms observed; 2 = only angular leaf spots; 3 = exclusive leaf blight, leaf wilt and defoliation, and gum exudation on the stem; 4 = exclusive leaf blight, defoliation, and die-back; and 5 = complete defoliation and stem die-back.
D. Cassava Anthracnose Disease (CAD) was visually characterized. A scale of 1 to 5 was used to rate symptoms: 1 = no symptoms observed; 2 = few shallow cankers on woody stems; 3 = many deep cankers on woody stems; 4 = many oval lesions on green stems; and 5 = many lesions on green stems and severe necrosis at leaf axils.

• Morphological and Agronomic traits evaluation

At harvest for seedling and clonal evaluation trials at 12 MAP, plant height (cm) was measured from the soil level to the highest apical point of the plant at harvest. Phenotypic assessments were performed on eight innermost plants per clone. Roots were separated from the vegetative harvestable biomass (leaves, stems, and the original planting stake) and independently weighed using a weighing balance. The fresh root yield and fresh foliage yield (kg plant⁻¹) were calculated. The harvest index (root biomass as a proportion of total biomass) was computed for each clone following the procedure outlined by Kawano (1990). Estimation of Dry Matter Content (DMC) (measured as a percentage) in the root samples was determined using the specific gravity method, as suggested by Kawano et al. (1987). The procedure included weighing of three (3) kg of roots with a weighing balance and then placing them in a nylon bag. The bag with roots was hung on a hanging balance of 6 kg capacity while suspending the bag in a tank with water, ensuring that the bag did not touch the bottom and that the roots were completely underwater. The weight of the water was then determined.(IITA, 1990)

Specific gravity = \frac{Weight in air}{Weight in air - Weight in water}

DMC = 158.3 specific gravity - 142.0

Data analysis

The number of progenies assessed differed across the families, making the dataset largely uneven. Summary statistics (average, range, variance, and skewness) for all traits were calculated for each family. The dataset for all the traits was subjected to a paired sample t-test using R. Inbreeding depression was estimated for plant height, vigor, fresh root yield, fresh foliage yield, harvest index, dry matter content, and disease data; cassava mosaic disease, cassava bacterial blight, and cassava anthracnose disease as a percentage of the S₁ average. ID was obtained based on the equation reported by Rojas et al. (2009):

ID = \frac{S_{0} mean - S_{1} mean}{S_{0} mean} \times 100

Inbreeding depression was assessed at the family level. The lower the ID value, the more severe the level of depression, indicating that the performance of the S₁ progenies is relatively close to that of the S₀ progenitor compared to the ID values that have higher depression.

The heterozygosity index was obtained using the following equation suggested by Scotti et al. (2000):

100 \times \frac{(S_{n} H - S_{n - x} H)}{S_{n - x} H}

Where S_n is the inbreeding generation in which the effect was calculated, S_n-x is the generation taken as a reference (the parental S₀), and H is the mean of heterozygosity loci. Data were analyzed using GenStat version 12.1 edition (2009).

Results

Origin of elite clones and size of their respective S₁ families

Table 1 presents the origin of the elite clones and the sizes of their respective S₁ families, which were evaluated in both seedling nursery and clonal evaluation trials. The selection of these families (TME 419, TMS 01/1371, TMS 30572, TMS 98/0002 and TMS 98/0505) for evaluation was based on the commercial significance of their progenitors in Africa. Specifically, family TME 419 started with 51 seeds, of which 33 genotypes were evaluated in seedling nurseries and 23 were evaluated in clonal evaluation trials. Similarly, for family TMS 01/1371, 79 seeds were produced, 63 were evaluated in the seedling nursery stage, and 35 were evaluated in the clonal stage. In the case of family TMS 30572, out of 111 seeds produced, 90 genotypes survived the seedling stage, and after the usual losses in the cloning process, 51 clones were evaluated in the clonal stage. For family TMS 98/0002, 44 out of the initial 64 seeds were evaluated in the seedling nursery and 24 in the clonal stage. Lastly, family TMS 98/0505 began with 68 seeds, but only 25 cloned genotypes were evaluated at the seedling stage, and only 14 cloned genotypes were evaluated at the clonal stage. These losses are typical and are also observed in the evaluation of the full-vigor germplasm. Despite these losses, the samples of S₁ genotypes representing the partially inbred populations were considered unbiased representations of all progenies that could be obtained from the self-pollination of the respective progenitors. This selection process ensures a fair representation of the evaluated progenies and allows for a comprehensive assessment of the performance and potential of partially inbred populations in breeding programs.

Table 1. Origin of the elite clones and sizes of their respective S₁ families evaluated in both trials.

Codes	Family name	Pedigree	No. of S₁ genotypes
Codes	Family name	Pedigree	SN	CET
GCO 219	TME 419	Landrace from Togo	33	23
GCO 223	TMS 01/1371	TMS 94/0561 X TMS 94/0263	63	35
GCO 228	TMS 30572	58308 X Branca de santa caterina	90	51
GCO 264	TMS 98/0002	97 DTP Rep 1	44	24
GCO 274	TMS 98/0505	97 DTP Rep 2	25	14
		Total	255	147

Effects of inbreeding depression on pests and diseases

Inbreeding depression (ID) is generally lower for pests and diseases than for complex traits such as yield. Inbreeding depression as a percentage of the performance from the S₀ generation measured in five S₁ families based on disease severity in seedling and clonal evaluation trials are presented in Tables 2 and 3, respectively. The disease data had negligible additive effects and had low ID values.

Table 2. Inbreeding depression measured in the five S₁ cassava families based on the disease severity at the seedling nursery trial.

Family	CMD			CBB			CAD
	S₀	S₁	ID	S₀	S₁	ID	S₀	S₁	ID
TME 419	1	2.41	-141	2	2.41	-20.5	2	2.31	-15.5
TMS 01/1371	1	2.15	-115	2	2.7	-35	2	2.23	-11.5
TMS 30572	2	1.99	0.5	3	3.21	-7	3	2.31	23
TMS 98/0002	1	2.1	-110	1	2.75	-175	1	2.13	-113
TMS 98/0505	1	2.06	-106	1	2.22	-122	1	2.17	-117
Average	1.2	2.14	-94.3	1.8	2.66	-71.9	1.8	2.23	-46.8

Table 3. Inbreeding depression measured in the five S₁ cassava families based on the disease severity at the clonal evaluation trial.

Family	CMD			CBB			CAD
	S₀	S₁	ID	S₀	S₁	ID	S₀	S₁	ID
TME 419	1	1.54	-53.91	2	2.57	-28.26	1.5	1.54	-2.9
TMS 01/1371	1	1.17	-16.57	2.5	2.27	9.14	1	1.53	-52.86
TMS 30572	1.25	1.97	-57.8	2	2.45	-22.55	1	1.55	-54.9
TMS 98/0002	1	1.39	-38.54	2	2.4	-19.79	1	1.54	-54.17
TMS 98/0505	1	1.39	-38.57	1.8	2.64	-46.83	1.5	1.54	-2.38
Average	1.05	1.49	-41.08	2.06	2.47	-21.66	1.2	1.54	-33.44

In the seedling evaluation trial, CMD had the lowest average ID value (-94.30%), which ranged from -141.00% (TME 419) to 0.50% (TMS 30572), followed by CBB with an average ID value of -71.90% and with a range from -175.00% (TMS 98/0002) to 7.00% (TMS 30572), followed by CAD with an average ID value of -46.80%, ranging from -117.00% (TMS 98/0505) to 23.00% (TMS 30572) (Table 2). In the clonal evaluation trial, CMD had the lowest average ID value (-41.08%), ranging from -57.80% (TMS 30572) to -16.57% (TMS 01/1371), followed by CAD with an average ID value of -33.44% and ranging from -54.90% (TMS 30572), followed by CBB with an average ID value of -21.66%, ranging from -46.83% (TMS 98/0505) to 9.14% (TMS 01/1371) (Table 3).

Phenotypic assessment of the families revealed some superior S₁ clones, which were found to substantially outperform their S₀ progenitors. In the case of cassava mosaic disease, cassava bacterial blight, and cassava anthracnose disease, no significant depression was observed (Tables 2 and 3).

Effects of inbreeding depression on agronomic traits

Agronomic traits revealed notable ID, with fresh root yield exhibiting the highest ID values at 86.55% and 53.05% at the seedling nursery and clonal evaluation stages, respectively (Tables 4 and 5). Other agronomic traits, such as fresh foliage yield and harvest index, also displayed considerable ID values (38% and 22%, respectively) at the clonal evaluation stage (Table 5). The effects of ID tended to be relatively higher for more complex traits given their polygenic character. Reduced heterozygosity associated with selfing (S₁) had a significant impact on fresh root yield and foliage, underscoring the relative importance of non-additive genetic effects in their inheritance. Some transgressive segregants were observed in the S₁.

Table 4. Inbreeding depression measured in five S₁ cassava (Manihot esculenta Crantz) families based on the yield traits in seedling nursery trial stage.

Family	Plant height (cm)			Fresh root yield (kg pl⁻¹)			Fresh foliage yield (kg pl⁻¹)			Harvest Index			Dry matter content (%)			Vigour
	S₀	S₁	ID	S₀	S₁	ID	S₀	S₁	ID	S₀	S₁	ID	S₀	S₁	ID	S₀	S₁	ID
TME 419	170	303.5	-78.53	40.8	3.33	91.84	11.8	4.28	63.73	0.78	0.46	40.7	33.18	29.39	11.41	5	3.63	27.5
TMS 01/1371	210	220.45	-4.98	17.8	4.56	74.38	6	3.45	42.5	0.75	0.55	26.46	25.93	24.68	4.81	3	3.09	-3.03
TMS 30572	200	213.33	-6.67	39.2	4.19	89.31	18.9	5.07	73.17	0.67	0.48	28.86	33.69	26.86	20.28	5	2.78	44.44
TMS 98/0002	146.5	207	-41.3	23.6	2.76	88.31	7.55	2.88	61.85	0.76	0.49	35.32	29.88	24.43	18.24	4	3.4	15
TMS 98/0505	250	240	4	48.6	5.4	88.89	19.2	1.8	90.63	0.72	0.75	-4.63	28.15	23.41	16.85	4	3.19	20.25
Average	195.3	236.86	-25.49	34	4.05	86.55	12.69	3.5	66.38	0.73	0.55	25.34	30.17	25.75	14.32	4.2	3.22	20.83

Table 5. Inbreeding depression measured in five S₁ cassava (Manihot esculenta Crantz) families based on the yield traits in clonal evaluation trial stage.

Family	Plant height (cm)			Fresh root yield (kg pl⁻¹)			Fresh foliage yield (kg pl⁻¹)			Harvest index			Dry matter content (%)			Vigour
	S₀	S₁	ID	S₀	S₁	ID	S₀	S₁	ID	S₀	S₁	ID	S₀	S₁	ID	S₀	S₁	ID
TME 419	231.2	244.52	-5.76	53.36	16.07	69.89	8.4	7.77	7.56	0.81	0.53	34.24	33.96	30.56	10.03	4.5	3.12	30.76
TMS 01/1371	191	198.83	-4.1	21.99	15.17	31	5.74	3.52	38.72	0.73	0.59	19.29	26.84	27.58	-2.75	2.5	2.46	1.71
TMS 30572	176.6	203.35	-15.15	46.28	18.16	60.76	15.72	5.12	67.44	0.66	0.59	10.42	29.3	26.51	9.52	4.2	2.25	46.47
TMS 98/0002	161.2	174.17	-8.04	27.99	10.92	60.98	6.44	4.37	32.19	0.77	0.51	34.34	29.3	27.31	6.8	3.8	2.7	28.95
TMS 98/0505	209.6	208.43	0.56	57.6	33.06	42.61	10.88	6.07	44.2	0.71	0.63	11.3	27.48	28.35	-3.18	3.6	2.4	33.2
Average	193.92	205.86	-6.5	41.44	18.68	53.05	9.44	5.37	38.02	0.74	0.57	21.92	29.38	28.06	4.08	3.72	2.59	28.22

Effects of inbreeding depression on morphological/architecture traits

As with pests and diseases, ID was lower than that of the complex traits. The average ID for vigour in the seedling evaluation trial was 20.83% ranging from -3.03% in family TMS 01/1371 to 44.44% in family TMS 30572 (Table 4) while in clonal evaluation trial, the average ID value for vigour was 28.22% ranging from 1.71% in family TMS 01/1371 to 46.47% in family TMS 30572 (Table 5). For plant height, the average ID observed at the seedling nursery and clonal evaluation stages were -25.49% and -6.50%, respectively, and all families produced S₁ genotypes with plant heights superior to those of their S₀ progenitor genotypes in both stages. This demonstrated that the performance of the progenies was relatively close to that of the S₀ progenitors. Based on these results, this set of traits appears to be less sensitive to inbreeding depression.

Paired (dependent) sample t-test: Seedling nursery stage vs. Clonal evaluation stage

A paired (dependent) sample t-test was used to compare the means of the seedling nursery and clonal evaluation stages to determine whether there was a statistically significant difference between these means. The results revealed a significant difference between the seedling nursery and clonal evaluation stages for all traits evaluated (Table 6). There was a significant difference in the CMD scores for the seedling nursery (M = 1.56, SD = 0.62) and clonal evaluation trials (M = 2.05, SD = 0.79); t (146) = 5.74, p = 0.000. For CBB, the clonal evaluation trial showed a significantly higher level of resistance (M = 2.44, SD = 0.39) than the seedling nursery stage (M = 3.01, SD = 0.70), t (146) = 8.25, p = 0.000. Similarly, the mean score for CAD at the clonal evaluation stage (M = 1.54, SD = 0.2) showed a higher level of resistance than the mean score at the seedling nursery stage (M = 2.26, SD = 0.54); t (146) = 15.72, p = 0.000.

Table 6. Paired (dependent) samples t-test analysis: Seedling nursery trial vs. Clonal evaluation trial stages.

Traits	Stages	Mean	SD	df	t-value	p
CMD	SN	1.56	0.62	146	5.74	0.000
CMD	CET	2.05	0.79	146	5.74	0.000
CBB	SN	3.01	0.7	146	8.25	0.000
CBB	CET	2.44	0.39	146	8.25	0.000
CAD	SN	2.26	0.54	146	15.72	0.000
CAD	CET	1.54	0.2	146	15.72	0.000
Vigour	SN	2.52	0.95	146	7.92	0.000
Vigour	CET	3.23	0.64	146	7.92	0.000
Plant height	SN	174.22	50.16	146	-4.87	0.000
Plant height	CET	204.44	57.06	146	-4.87	0.000
FFY	SN	1.44	1.81	146	-8.42	0.000
FFY	CET	5.17	5.03	146	-8.42	0.000
HI	SN	0.49	0.19	146	-14.17	0.000
HI	CET	0.57	0.15	146	-14.17	0.000
FRY	SN	1.53	1.78	146	-3.72	0.000
FRY	CET	17.28	13.22	146	-3.72	0.000
DMC	SN	29.89	6.75	146	3.48	0.001
DMC	CET	27.54	5.24	146	3.48	0.001

More vigorous plants were observed at the clonal evaluation stage (M = 3.23, SD = 0.64) than at the seedling nursery stage (M = 2.52, SD = 0.95; t (146) = 7.92, p = 0.000). There was a significant difference between the heights of plants at the seedling nursery (M = 174.22, SD = 50.16) and clonal evaluation stages (M = 204.44, SD = 57.06); t (146) = -4.87, p = 0.000.

There was a significant improvement in fresh foliage yield from the seedling nursery stage (M = 1.44, SD = 1.81) to the clonal evaluation stage (M = 5.17, SD = 5.03); t (146) = -8.42, p = 0.000. The result also showed that fresh root yield was significantly higher at the clonal evaluation stage (M = 17.28, SD = 13.22) than at the seedling nursery stage (M = 1.53, SD = 1.78); t (146) = -3.72, p = 0.000. The dependent sample t-test showed that the seedling nursery stage (M = 0.49, SD = 0.19) had a lower mean harvest index value than the clonal evaluation stage (M = 0.57, SD = 0.15), t (146) = -14.17, p = 0.000. The clonal evaluation trial showed significantly lower root dry matter content (M = 27.54, SD = 5.24) than the seedling nursery stage (M = 29.89, SD = 6.75); t (146) = 3.48, p = 0.001.

SNP estimates for heterozygosity

Using SNPs molecular markers for heterozygosity, the S₁ generation showed a good proportion of heterozygotes at several loci across the genome, indicating that heterozygosity is very high in cassava, and several selfing cycles may be required to reduce it drastically. The heterozygosity indices of S₁ progenies are shown in Figure 1. SNP analysis revealed percentage heterozygosity between 12% and 65% in the population. The average heterozygosity reported for the S₁ families is shown in Table 7. The results showed that estimates of the heterozygosity index tended to increase and improve as a greater number of SNP markers were used. This suggests that an optimum number of SNPs should be determined to improve the precision of heterozygosity estimates in cassava. Figure 2 shows the distribution of S₁ progenies based on their percentage heterozygosity estimated using SNP markers. The findings also demonstrated that a majority of the progenies, specifically over 55%, exhibited heterozygosity values below 25%. This suggests that there are quality materials to pass on to the next inbreeding generation, which will likely result in less heterozygosity in the subsequent breeding generation.

Figure 1. The effect of number of SNPs on the heterozygosity index with the exception of TMS 98/0002.

Table 7. Heterozygosity index measured in five S₁ cassava (Manihot esculenta Crantz) families.

Family	S₀	S₁	Heterozygosity index
TME 419	39.5	31	-21.32
TMS 01/1371	27	24.4	-9.69
TMS 30572	39.2	30.3	-22.87
TMS 98/0002	7.32	23.3	218.02
TMS 98/0505	46	26.2	-43.13
Average	31.8	27	24.21

Figure 2. Distribution of S1 progenies (except TMS 98/0002) based on their percentage heterozygosity using SNPs markers.

Discussion

Phenotypic measurements of key breeding traits were performed during the seedling and clonal evaluation trials. This study explored SNP markers to assess the heterozygosity of the top five elite clones that have been extensively used as parents in cassava breeding programs.

The inbreeding depression for fresh root yield, which is a very complex trait (involving many genes), was found to be higher than the values obtained for other traits evaluated in this study. These results suggest that a high ID was more likely for traits that were more quantitatively controlled (e.g., root yield) than traits controlled by fewer genes, for example, plant height. This agrees with the findings of Castro et al. (2006).

In the case of cassava mosaic disease, cassava bacterial blight, and cassava anthracnose disease, little depression was observed. This was probably because of additive genetic effects, in addition to the fact that pests and disease resistance are often affected by a few genes (including dominant or major gene effects). For example, previous molecular studies indicated that CMD resistance in two of the varieties, TMS98/0505 and TMS98/0002, used as parents, is mainly controlled by a single dominant gene resistance (Okogbenin et al., 2013).

These results suggest that breeders can explore inbreeding for these traits in the early stages of the selfing cycle within the breeding scheme, potentially enhancing their genetic progress. Some previous studies in cassava have reported disease resistance to CMD and CBB as being quantitatively controlled, with some being recessive. Furthermore, the low ID for pests and diseases shows that traits that are strongly influenced by additive genetic variance stand to benefit most, leading to genetic gain. Additionally, useful beneficial recessive traits can be effectively explored while simultaneously selecting undesirable recessive traits.

In general, the inbreeding depression values were higher in the seedling evaluation trial than in the clonal evaluation trial for fresh root yield, fresh foliage yield, harvest index, and dry matter content. The use of seeds versus stem cuttings may have influenced the results. In the seedling trial, cassava was raised from seeds, whereas stem cuttings were used as planting materials in the clonal trial. Seeds typically produce plants with fewer and smaller roots than those raised from stem cuttings (clonal stage) (FAO, 1977). Food reserves in stem cuttings aid in rapid establishment and improved performance. Therefore, it is important to explore later generations to evaluate inbreeding depression for these traits.

A paired samples t-test was performed to determine whether there was a statistically significant difference between seedling nursery and clonal evaluation stages for all traits evaluated. The results revealed a significant difference between the seedling nursery and clonal evaluation stages for all traits evaluated. The expression of the disease traits (cassava mosaic disease, cassava bacterial blight, and cassava anthracnose disease) was more severe at the seedling nursery stage than at the clonal evaluation stage. Likewise, morphological traits (vigor and plant height) performed better at the clonal evaluation stage than at the seedling stage. This could be because during the process of moving from the seedling evaluation stage to the clonal evaluation stage, genotypes with poor vigor were eliminated, while vigorous genotypes were selected for further testing. For yield traits, such as fresh foliage yield, fresh root yield, and harvest index, the use of stem cuttings may have provided better results in the clonal evaluation stage. The dry matter content was significantly lower at the clonal evaluation stage than at the seedling nursery stage. At the seedling nursery stage, the genotypes mainly had fibrous roots and a higher dry matter content than at the clonal evaluation, where tuberous roots and a lower dry matter content were observed.

Cassava has been demonstrated to be a highly heterozygous species (Kawano et al., 1978; Pujol et al., 2005). Inbreeding depression, such as that observed in this study, was therefore expected. ID is the result of a reduction in heterozygosity levels in loci with dominant gene effects and because of the increased frequency of expression of unfavorable alleles (Falconer, 1981; Miranda-Filho, 1999, Vencovsky and Barriga, 1992). Inbred lines are more suitable for breeding and genetic research due to their lack of dominance effect and reduced genetic load (undesirable alleles), making them superior parents.

Five S₁ individuals, one genotype each from the families of TMS 98/0505, TME 419, and TMS 30572 and two genotypes from the family of TMS 01/1371 showing relatively low heterozygosity based on SNP data, were selected as parents for further selfing to assess ID. Therefore, these genotypes were further advanced to generate an S₂ population for further study.

Conclusion

The impact of inbreeding on pests, diseases, and morphological and architectural traits in the S₁ generation was non-severe. Notably, the presence of transgressive segregants in the population for key strategic traits of the crop is indicative of favorable allele combinations at the S₁ stage. Therefore, the results demonstrate that inbreeding can be strategically explored in breeding for these traits to increase genetic gain and possibly identify recessive traits. At the early stages of the cassava breeding scheme, most key traits, including disease resistance, were not severely affected at S₁. This presents an opportunity to identify major or a few genes associated with disease resistance and to fix them in early generations to generate valuable parent stocks. These parent stocks can then be used in crosses, enabling the exploration of both additive and non-additive variances for polygenic traits in later breeding cycles. Moreover, the strategic use of inbreeding allows for early selection of productivity traits once genetic stocks become available. This can significantly shorten the breeding cycle, especially when appropriate technologies are employed to rapidly multiply selected genotypes in larger yield trials. In summary, our findings suggest that inbreeding can be a valuable tool in cassava breeding for specific traits, offering potential benefits in accelerating genetic progress and identifying essential recessive traits. By incorporating inbreeding strategies and leveraging advanced technologies, cassava breeding programs can make significant strides in developing improved varieties that address the challenges of pests, diseases, and productivity in this vital crop.

Data availability statement

The datasets presented in this study can be found in the figshare repository.

1. SNPs estimates for heterozygosity , DOIs: https://doi.org/10.6084/m9.figshare.26312278.v1 (Jiwuba, 2024b)
This project contains the following underlying data:
- • S1 SNPS.xlsx
2. Inbreeding depression seedling and clonal evaluation trials.xlsx, Doi: https://doi.org/10.6084/m9.figshare.26317900 (Jiwuba, 2024a)
This project contains the following underlying data:
- • inbreeding depression seedling and clonal evaluation trials.xlsx

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

Acknowledgments

Special thanks to the technical team working at NRCRI, Umudike, for their assistance during the field activities and data collection.

References

Castro AM, Perez JP, Fregene M, et al.: Use of Molecular markers to estimate level of heterozygosity in selfed lines of Cassava (Manihot esculenta Crantz) . Project IP3, Improved cassava for the developing world. CIAT Annual report 2006. Cali, Colombia: Centro internacional de Agricultura Tropical (CIAT); 2006.
Ceballos H, Iglesias CA, Perez JC, et al.: Cassava breeding: Opportunities and challenges. Plant Mol. Biol. 2004; 56: pp. 503–516.
Ceballos H, Fregene M, Perez JC, et al.: Cassava genetic improvement. Breeding major food staples. 2007; 965–991.
Dellarporta SL, Wood J, Hicks JR: A plant DNA minipreparation: version II. Plant Mol. Biol. Report. 1983; 1: 19–21.
Falconer DS: Introduction to Quantitative Genetics. 2nd ed.New York, USA: Longman; 1981; p. 400.
FAO: Cassava Processing. Document prepared by M.R. Grace. FAO Plant Production and Protection Series No.3.1977.
Federer WT: Augmented (or hoonuiaku) designs. Hawaiian Planters’ Record LV. 1956; 2: 191–208.
GenStat: GenStat for Windows. 12th ed.Hemel Hempstead, UK: VSN International; 2009. Web page: GenStat.co.uk.
IITA: Cassava in tropical Africa. A reference manual. Ibadan, Nigeria: International Institute of Tropical Agriculture; 1990.
Jiwuba L: inbreeding depression seedling and clonal evaluation trials.xlsx (Version 1). figshare. 2024a. Publisher Full Text
Jiwuba L: S1 SNPS.xlsx (Version 1). figshare. 2024b. Publisher Full Text
Kawano K, Amaya A, Daza P, et al.: Factors affecting efficiency of hybridization and selection in cassava (Manihot esculenta Crantz). Crop Sci. 1978; 18: 373–376.
Kawano K: Harvest index and evolution of major food crops cultivars in the tropics. Euphytica. 1990; 46: 195–202.
Kawano K, Gonçalves FW, Fukuda M, et al.: Genetics and environmental effects on dry matter content of cassava roots. Crop Sci. 1987; 27: 69–74.
Miranda-Filho JB: Inbreeding depression and heterosis. The genetic exploitation of heterosis in crops. Coors JG, Pandey S, editors. Madison WI: ASA; 1999; pp. 69–81.
Okogbenin E, Moreno I, Tomkins J, et al.: Marker-assisted breeding for cassava mosaic disease resistance. Translational genomics for crop breeding: biotic stress. 2013; 1: 291–325.
Pujol BP, David P, McKey D: Microevolution in agricultural environments: How a traditional Amerindian farming practice favours heterozygosity in cassava (Manihot esculenta Crantz, Euphorbiaceae). Ecol. Lett. 2005; 8: 138–147.
Rojas MC, Perez JC, Cellabos H, et al.: Analysis of inbreeding depression in eight S₁cassava families. Crop Sci. 2009; 49: 543–548.
Scotti C, Pupilli F, Salvi S, et al.: Variation in vigor and in RFLP-estimated heterozygosity by selfing tetraploid alfalfa: new perspectives for the use of selfing in alfalfa breeding. Theor. Appl. Genetics. 2000; 101: 120–125.
Vencovsky R, Barriga P: Genética Biométrica no Fitomelhoramento. Ribeirão Preto, Brazil: Sociedade Brasileira de Genética; 1992; p. 486.

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Author details Author details

¹ Biotechnology, NRCRI, National Root Crops Research Institute, Umudike, 7006, Nigeria
² Department of Plant breeding and genetics, Cornell University, Ithaca, New York, 14850, USA
³ NABDA, National Biotechnology Development Agency, Airport Road, Abuja, Nigeria
⁴ College of Crop and Soil Science, Michael Okpara Univeristy of Agriculture, Umudike, Nigeria
⁵ Biosciences, International Institute of Tropical Agriculture, Ibadan, Oyo, Nigeria
⁶ AATF, African Agricultural Technology Foundation, Nairobi, Kenya

Lydia Jiwuba
Roles: Conceptualization, Data Curation, Formal Analysis, Investigation, Methodology, Resources, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

Alex Ogbonna
Roles: Data Curation, Formal Analysis, Writing – Review & Editing

Ugochukwu Ikeogu
Roles: Formal Analysis, Methodology, Resources, Writing – Review & Editing

Favour Okeakpu
Roles: Methodology, Resources, Writing – Review & Editing

Kenneth Eluwa
Roles: Methodology, Resources

Eunice Ekaette
Roles: Methodology, Resources, Writing – Review & Editing

Damian Njoku
Roles: Methodology, Writing – Review & Editing

Peter Okocha
Roles: Supervision, Writing – Review & Editing

Chiedozie Egesi
Roles: Investigation, Resources, Supervision, Visualization, Writing – Review & Editing

Emmanuel Okogbenin
Roles: Conceptualization, Funding Acquisition, Investigation, Methodology, Project Administration, Resources, Supervision, Validation, Visualization, Writing – Review & Editing

Competing interests

No competing interests were disclosed.

Grant information

This study was conducted with funding support from the Generation Challenge Program (GCP) (grant number G7010.01.05) and the National Root Crops Research Institute (NRCRI), Umudike, Nigeria .
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Jiwuba L, Ogbonna A, Ikeogu U et al. Analysis of inbreeding depression in five S₁ Cassava families of elite varieties at the seedling nursery and clonal evaluation trial stages. [version 1; peer review: 1 approved, 1 approved with reservations]. F1000Research 2024, 13:966 (https://doi.org/10.12688/f1000research.153922.1)

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Reviewer Report 13 Feb 2025

Karoline Leonard Sichalwe, Makerere University, Kampala, Uganda; Research and Innovation, Tanzania Agricultural Research Institute, Dodoma, Tanzania

Approved with Reservations

https://doi.org/10.5256/f1000research.168880.r362241

General overview
The article presents a well-structured and relevant study on inbreeding depression in cassava, a highly heterozygous and economically important crop. It effectively combines field trials and genotyping to assess the impact of inbreeding on key traits, including ... Continue reading

General overview
The article presents a well-structured and relevant study on inbreeding depression in cassava, a highly heterozygous and economically important crop. It effectively combines field trials and genotyping to assess the impact of inbreeding on key traits, including yield, disease resistance, and morphological characteristics. The study is valuable for cassava breeding programs, as it highlights the potential benefits of controlled inbreeding, such as fixing beneficial alleles and eliminating deleterious ones. Additionally, the identification of transgressive segregants suggests promising genetic improvements. The inclusion of both seedling and clonal evaluation trials provides a comprehensive assessment, strengthening the study’s applicability in breeding strategies. However, there are some improvements which can be made to make it better.

Introduction

While this context is useful, it could be more focused and directly related to the study’s aims. more specific discussion of the relevance of inbreeding depression in cassava
The concept of inbreeding depression is introduced and explained twice, once early on and again later in the paragraph. This repetition dilutes the impact of the key ideas. Streamline the explanation of inbreeding depression into a single concise paragraph
The introduction mentions inbreeding depression and cassava's genetic variability but doesn't clearly indicate how the study contributes to this body of research. Clearly state the study’s specific aims, such as: "This study aims to quantify inbreeding depression in five S1 cassava families at both the seedling nursery and clonal evaluation trial stages.
The citations to previous studies are scattered and appear somewhat disjointed
The potential benefits of inbreeding could be shortened to maintain the focus on the study’s goals. Condense the explanation of inbreeding benefits, focusing more on how it applies specifically to cassava improvement. For instance: "Inbreeding in cassava may offer benefits such as eliminating harmful recessive genes and accumulating superior traits, leading to improved productivity and genetic stability.
The last sentence feels a bit rushed and vague. It mentions the potential for inbreeding to enhance cassava breeding programs but doesn't succinctly summarize how the study will contribute to this goal . I suggest to end with a more direct and impactful statement, such as: "This study aims to provide critical insights into inbreeding depression in cassava, enabling more effective utilization of inbreeding in breeding programs and ultimately enhancing cassava's role in sustainable agricultural development."

Methodology

The information about the study site (NRCRI Umudike) is very detailed, but it may be unnecessary to include such specific geographic data (latitude, longitude, altitude) unless it directly influences the study. It could be streamlined for clarity. Consider focusing more on the environmental conditions that impact the experiment, such as rainfall, temperature, and humidity, and how they relate to cassava growth. For example: "The field experiment was conducted at the National Root Crops Research Institute, Umudike, Nigeria, where environmental conditions such as annual rainfall (2,166.3 mm), temperature (22°C - 32°C), and high relative humidity (80–90%) are optimal for cassava cultivation”.
On genotype selection, that the genotypes were not randomly selected because of their wide adoption by farmers, but this reasoning could be clarified. It’s not clear how their selection for favorable morphological and agronomic attributes directly ties into the objectives of the study. Clarify the traits selected for to show the rationale behind the selection which makes them ideal candidates for evaluating the impacts of inbreeding depression in elite cassava breeding programs
The process of self-pollination is described in a somewhat disjointed way. It mentions isolating the plots and securing pollinated flowers but could be more cohesive. Consider re-phrasing to make it more smoothly “ "To ensure self-pollination, the plots were isolated from neighboring cassava plots by 100 meters, and flowers were pollinated manually. Pollination bags were used to protect the flowers from external pollen sources. The bags were removed the following day, and the mature fruits were harvested 90 days after pollination."
The process of establishing the seedling evaluation trial is clear but could benefit from a bit more focus on why certain practices (like the dormancy period or specific soil mixture) are essential for cassava growth. Consider provide a brief explanation of why certain procedures were used: "The harvested S1 seeds underwent a two-month dormancy period, which is necessary to break seed dormancy and encourage uniform germination. They were then sown in sterilized soil with a loamy-sandy mixture (2:1) to ensure proper root development and nutrient uptake."
The narrative does not specify the duration of the clonal evaluation trial or what specific measurements/parameters were evaluated (e.g., yield, disease resistance, etc.), which would be important for readers to understand the scope of the trial. Consider adding relevant details to round out the process: "The clonal evaluation trial lasted for 12 months, during which the genotypes were evaluated for key agronomic traits such as yield, disease resistance, and growth performance."

Discussion

The introduction of new ideas is abrupt, and some paragraphs cover multiple aspects without a clear topic sentence. Consider writing clear topic sentence that summarizes the key point, making it easier for the reader to follow the discussion.
The study mentions that SNP markers were used to assess heterozygosity but does not explain how this directly relates to inbreeding depression. Briefly explain why SNP markers are relevant in identifying ID patterns or why heterozygosity was a key focus in relation to inbreeding effects.
The explanation of why inbreeding depression is higher for fresh root yield than for other traits is overly simplistic. The discussion mentions that quantitative traits (such as root yield) have a higher ID than traits controlled by fewer genes (such as plant height), but this could be elaborated upon with more genetic reasoning. Include a more detailed explanation of polygenic inheritance and why ID tends to be stronger in such traits due to the cumulative effects of deleterious recessive alleles.
The section on disease resistance mentions that ID was low due to additive genetic effects but then states that some previous studies reported disease resistance as quantitatively controlled. This inconsistency makes it unclear whether resistance is mostly additive or influenced by multiple genes. Clarify whether disease resistance in cassava is predominantly controlled by major genes or polygenic effects and how this impacts inbreeding depression.
The section on the differences between seedling and clonal trials attributes better performance in the clonal stage to the use of stem cuttings but does not discuss potential confounding factors such as selection pressure or environmental influences. Improve by acknowledging other factors that may contribute to differences in performance between the seedling and clonal evaluation stages beyond just the planting material.
While a paired samples t-test is mentioned, there is no detail on the statistical significance (p-values, effect sizes) or discussion on potential sources of variation. Improve by report p-values or confidence intervals for key findings and discuss potential limitations, such as environmental effects or sample size constraints.
Consider concluding statement on the broader implications of the findings. Improve by a concise statement summarizing the study's significance for cassava breeding programs and how inbreeding strategies can be optimized.

Conclusion

The statement about "transgressive segregants" suggesting favorable allele combinations is not fully explained. It assumes that transgressive segregation always leads to beneficial traits, which is not necessarily true. Improve by clarifying whether these transgressive segregants were beneficial or detrimental for specific traits. Were they more vigorous, resistant to diseases, or higher yielding? Providing examples would strengthen the argument.
The claim that early inbreeding allows fixation of disease resistance genes is not well justified. If disease resistance is controlled by major genes, how does inbreeding help fix them, and what evidence supports this?. Consider providing genetic or molecular evidence (e.g., previous studies or marker data) that suggests disease resistance genes can be efficiently fixed through inbreeding in cassava.
End with a concise statement highlighting the key findings and specific implications for breeding, such as: "Our findings indicate that inbreeding can be leveraged to fix favorable alleles while minimizing genetic load, particularly for disease resistance and morphological traits, thereby accelerating cassava improvement”

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

I cannot comment. A qualified statistician is required.
Are all the source data underlying the results available to ensure full reproducibility?

No source data required
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Agricultural researcher specializing in variety improvement, with expertise in breeding new crop varieties using both conventional and biotechnology approaches. Skilled in seed conservation, seed systems development, and enhancing genetic diversity for improved agricultural productivity and sustainability.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

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Reviewer Report 17 Oct 2024

Charles Orek, Murang'a University of Technology, Murang'a, Kenya

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https://doi.org/10.5256/f1000research.168880.r327456

Exploitation of inbreeding for improved cassava performance is an exciting but relatively unexplored field of research. This study is thus foundational. My brief comments on each section that the authors can improved is as below:

1) Introduction ... Continue reading

Exploitation of inbreeding for improved cassava performance is an exciting but relatively unexplored field of research. This study is thus foundational. My brief comments on each section that the authors can improved is as below:

1) Introduction / background is extremely brief. The authors can add more information for proper justification of the study.
2) Seed production in cassava takes a long time. The authors can indicate how long it took for the seeds to mature post-pollination
3) Some references are very old (e.g. Federer, 1956; Dellarporta et al. 1983; Kawano et al. 1987; IITA, 1990; Kawano, 1990; Scotti et al. 2000, FAO, 1977 among others. The authors can replace these with more recent references to indicate that they are up to date with recent development in this particular field of research. Unless no recent research has been previously done...…..then the old references can make sense.
4) The authors have not indicated references for the Pests and Diseases measurements. For examples references for the methods the authors adopted in scoring of CMD, CBB and CAD are lacking. The sources of these methods should be added. Unless the authors are the ones who came up with the scoring methods.
5) "This agrees with the findings of Castro et al. (2006)". Perhaps the authors can indicate / report a one-sentence summary of the actual findings from Castro at al. 2006.
6) "Some previous studies in cassava have reported disease resistance to CMD and CBB as being quantitatively controlled, with some being recessive". The authors can provide references for these studies for ease of confirmation of this point in the discussion.

NB: This is a good research initiative and more should be done in exploitation inbreeding for better cassava performance.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Yes
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests: No competing interests were disclosed.

Reviewer Expertise: Application of biotechnological tools for biotic and abiotic stress resistance in cassava and other root crops

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

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Charles Orek, Murang'a University of Technology, Murang'a, Kenya
Karoline Leonard Sichalwe, Makerere University, Kampala, Uganda; Tanzania Agricultural Research Institute, Dodoma, Tanzania

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Karoline Leonard Sichalwe, Makerere University, Kampala, Uganda; Research and Innovation, Tanzania Agricultural Research Institute, Dodoma, Tanzania

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Approved With Reservations

General overview
The article presents a well-structured and relevant study on inbreeding depression in cassava, a highly heterozygous and economically important crop. It effectively combines field trials and genotyping to assess the impact of inbreeding on key traits, including yield, disease resistance, and morphological characteristics. The study is valuable for cassava breeding programs, as it highlights the potential benefits of controlled inbreeding, such as fixing beneficial alleles and eliminating deleterious ones. Additionally, the identification of transgressive segregants suggests promising genetic improvements. The inclusion of both seedling and clonal evaluation trials provides a comprehensive assessment, strengthening the study’s applicability in breeding strategies. However, there are some improvements which can be made to make it better.

Introduction

While this context is useful, it could be more focused and directly related to the study’s aims. more specific discussion of the relevance of inbreeding depression in cassava
The concept of inbreeding depression is introduced and explained twice, once early on and again later in the paragraph. This repetition dilutes the impact of the key ideas. Streamline the explanation of inbreeding depression into a single concise paragraph
The introduction mentions inbreeding depression and cassava's genetic variability but doesn't clearly indicate how the study contributes to this body of research. Clearly state the study’s specific aims, such as: "This study aims to quantify inbreeding depression in five S1 cassava families at both the seedling nursery and clonal evaluation trial stages.
The citations to previous studies are scattered and appear somewhat disjointed
The potential benefits of inbreeding could be shortened to maintain the focus on the study’s goals. Condense the explanation of inbreeding benefits, focusing more on how it applies specifically to cassava improvement. For instance: "Inbreeding in cassava may offer benefits such as eliminating harmful recessive genes and accumulating superior traits, leading to improved productivity and genetic stability.
The last sentence feels a bit rushed and vague. It mentions the potential for inbreeding to enhance cassava breeding programs but doesn't succinctly summarize how the study will contribute to this goal . I suggest to end with a more direct and impactful statement, such as: "This study aims to provide critical insights into inbreeding depression in cassava, enabling more effective utilization of inbreeding in breeding programs and ultimately enhancing cassava's role in sustainable agricultural development."

Methodology

The information about the study site (NRCRI Umudike) is very detailed, but it may be unnecessary to include such specific geographic data (latitude, longitude, altitude) unless it directly influences the study. It could be streamlined for clarity. Consider focusing more on the environmental conditions that impact the experiment, such as rainfall, temperature, and humidity, and how they relate to cassava growth. For example: "The field experiment was conducted at the National Root Crops Research Institute, Umudike, Nigeria, where environmental conditions such as annual rainfall (2,166.3 mm), temperature (22°C - 32°C), and high relative humidity (80–90%) are optimal for cassava cultivation”.
On genotype selection, that the genotypes were not randomly selected because of their wide adoption by farmers, but this reasoning could be clarified. It’s not clear how their selection for favorable morphological and agronomic attributes directly ties into the objectives of the study. Clarify the traits selected for to show the rationale behind the selection which makes them ideal candidates for evaluating the impacts of inbreeding depression in elite cassava breeding programs
The process of self-pollination is described in a somewhat disjointed way. It mentions isolating the plots and securing pollinated flowers but could be more cohesive. Consider re-phrasing to make it more smoothly “ "To ensure self-pollination, the plots were isolated from neighboring cassava plots by 100 meters, and flowers were pollinated manually. Pollination bags were used to protect the flowers from external pollen sources. The bags were removed the following day, and the mature fruits were harvested 90 days after pollination."
The process of establishing the seedling evaluation trial is clear but could benefit from a bit more focus on why certain practices (like the dormancy period or specific soil mixture) are essential for cassava growth. Consider provide a brief explanation of why certain procedures were used: "The harvested S1 seeds underwent a two-month dormancy period, which is necessary to break seed dormancy and encourage uniform germination. They were then sown in sterilized soil with a loamy-sandy mixture (2:1) to ensure proper root development and nutrient uptake."
The narrative does not specify the duration of the clonal evaluation trial or what specific measurements/parameters were evaluated (e.g., yield, disease resistance, etc.), which would be important for readers to understand the scope of the trial. Consider adding relevant details to round out the process: "The clonal evaluation trial lasted for 12 months, during which the genotypes were evaluated for key agronomic traits such as yield, disease resistance, and growth performance."

Discussion

The introduction of new ideas is abrupt, and some paragraphs cover multiple aspects without a clear topic sentence. Consider writing clear topic sentence that summarizes the key point, making it easier for the reader to follow the discussion.
The study mentions that SNP markers were used to assess heterozygosity but does not explain how this directly relates to inbreeding depression. Briefly explain why SNP markers are relevant in identifying ID patterns or why heterozygosity was a key focus in relation to inbreeding effects.
The explanation of why inbreeding depression is higher for fresh root yield than for other traits is overly simplistic. The discussion mentions that quantitative traits (such as root yield) have a higher ID than traits controlled by fewer genes (such as plant height), but this could be elaborated upon with more genetic reasoning. Include a more detailed explanation of polygenic inheritance and why ID tends to be stronger in such traits due to the cumulative effects of deleterious recessive alleles.
The section on disease resistance mentions that ID was low due to additive genetic effects but then states that some previous studies reported disease resistance as quantitatively controlled. This inconsistency makes it unclear whether resistance is mostly additive or influenced by multiple genes. Clarify whether disease resistance in cassava is predominantly controlled by major genes or polygenic effects and how this impacts inbreeding depression.
The section on the differences between seedling and clonal trials attributes better performance in the clonal stage to the use of stem cuttings but does not discuss potential confounding factors such as selection pressure or environmental influences. Improve by acknowledging other factors that may contribute to differences in performance between the seedling and clonal evaluation stages beyond just the planting material.
While a paired samples t-test is mentioned, there is no detail on the statistical significance (p-values, effect sizes) or discussion on potential sources of variation. Improve by report p-values or confidence intervals for key findings and discuss potential limitations, such as environmental effects or sample size constraints.
Consider concluding statement on the broader implications of the findings. Improve by a concise statement summarizing the study's significance for cassava breeding programs and how inbreeding strategies can be optimized.

Conclusion

The statement about "transgressive segregants" suggesting favorable allele combinations is not fully explained. It assumes that transgressive segregation always leads to beneficial traits, which is not necessarily true. Improve by clarifying whether these transgressive segregants were beneficial or detrimental for specific traits. Were they more vigorous, resistant to diseases, or higher yielding? Providing examples would strengthen the argument.
The claim that early inbreeding allows fixation of disease resistance genes is not well justified. If disease resistance is controlled by major genes, how does inbreeding help fix them, and what evidence supports this?. Consider providing genetic or molecular evidence (e.g., previous studies or marker data) that suggests disease resistance genes can be efficiently fixed through inbreeding in cassava.
End with a concise statement highlighting the key findings and specific implications for breeding, such as: "Our findings indicate that inbreeding can be leveraged to fix favorable alleles while minimizing genetic load, particularly for disease resistance and morphological traits, thereby accelerating cassava improvement”

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Partly
Are sufficient details of methods and analysis provided to allow replication by others?

Partly
If applicable, is the statistical analysis and its interpretation appropriate?

I cannot comment. A qualified statistician is required.
Are all the source data underlying the results available to ensure full reproducibility?

No source data required
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Agricultural researcher specializing in variety improvement, with expertise in breeding new crop varieties using both conventional and biotechnology approaches. Skilled in seed conservation, seed systems development, and enhancing genetic diversity for improved agricultural productivity and sustainability.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

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Reviewer Report

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17 Oct 2024 | for Version 1

Charles Orek, Murang'a University of Technology, Murang'a, Kenya

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Exploitation of inbreeding for improved cassava performance is an exciting but relatively unexplored field of research. This study is thus foundational. My brief comments on each section that the authors can improved is as below:

1) Introduction / background is extremely brief. The authors can add more information for proper justification of the study.
2) Seed production in cassava takes a long time. The authors can indicate how long it took for the seeds to mature post-pollination
3) Some references are very old (e.g. Federer, 1956; Dellarporta et al. 1983; Kawano et al. 1987; IITA, 1990; Kawano, 1990; Scotti et al. 2000, FAO, 1977 among others. The authors can replace these with more recent references to indicate that they are up to date with recent development in this particular field of research. Unless no recent research has been previously done...…..then the old references can make sense.
4) The authors have not indicated references for the Pests and Diseases measurements. For examples references for the methods the authors adopted in scoring of CMD, CBB and CAD are lacking. The sources of these methods should be added. Unless the authors are the ones who came up with the scoring methods.
5) "This agrees with the findings of Castro et al. (2006)". Perhaps the authors can indicate / report a one-sentence summary of the actual findings from Castro at al. 2006.
6) "Some previous studies in cassava have reported disease resistance to CMD and CBB as being quantitatively controlled, with some being recessive". The authors can provide references for these studies for ease of confirmation of this point in the discussion.

NB: This is a good research initiative and more should be done in exploitation inbreeding for better cassava performance.

Is the work clearly and accurately presented and does it cite the current literature?

Yes
Is the study design appropriate and is the work technically sound?

Yes
Are sufficient details of methods and analysis provided to allow replication by others?

Yes
If applicable, is the statistical analysis and its interpretation appropriate?

Yes
Are all the source data underlying the results available to ensure full reproducibility?

Yes
Are the conclusions drawn adequately supported by the results?

Yes

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Application of biotechnological tools for biotic and abiotic stress resistance in cassava and other root crops

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Respond to this report

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[1] Castro AM, Perez JP, Fregene M, et al.: Use of Molecular markers to estimate level of heterozygosity in selfed lines of Cassava (Manihot esculenta Crantz) . Project IP3, Improved cassava for the developing world. CIAT Annual report 2006. Cali, Colombia: Centro internacional de Agricultura Tropical (CIAT); 2006.

[2] Ceballos H, Iglesias CA, Perez JC, et al.: Cassava breeding: Opportunities and challenges. Plant Mol. Biol. 2004; 56: pp. 503–516.

[3] Ceballos H, Fregene M, Perez JC, et al.: Cassava genetic improvement. Breeding major food staples. 2007; 965–991.

[4] Dellarporta SL, Wood J, Hicks JR: A plant DNA minipreparation: version II. Plant Mol. Biol. Report. 1983; 1: 19–21.

[5] Falconer DS: Introduction to Quantitative Genetics. 2nd ed.New York, USA: Longman; 1981; p. 400.

[6] FAO: Cassava Processing. Document prepared by M.R. Grace. FAO Plant Production and Protection Series No.3.1977.

[7] Federer WT: Augmented (or hoonuiaku) designs. Hawaiian Planters’ Record LV. 1956; 2: 191–208.

[8] GenStat: GenStat for Windows. 12th ed.Hemel Hempstead, UK: VSN International; 2009. Web page: GenStat.co.uk.

[9] IITA: Cassava in tropical Africa. A reference manual. Ibadan, Nigeria: International Institute of Tropical Agriculture; 1990.

[10] Jiwuba L: inbreeding depression seedling and clonal evaluation trials.xlsx (Version 1). figshare. 2024a. Publisher Full Text

[11] Jiwuba L: S1 SNPS.xlsx (Version 1). figshare. 2024b. Publisher Full Text

[12] Kawano K, Amaya A, Daza P, et al.: Factors affecting efficiency of hybridization and selection in cassava (Manihot esculenta Crantz). Crop Sci. 1978; 18: 373–376.

[13] Kawano K: Harvest index and evolution of major food crops cultivars in the tropics. Euphytica. 1990; 46: 195–202.

[14] Kawano K, Gonçalves FW, Fukuda M, et al.: Genetics and environmental effects on dry matter content of cassava roots. Crop Sci. 1987; 27: 69–74.

[15] Miranda-Filho JB: Inbreeding depression and heterosis. The genetic exploitation of heterosis in crops. Coors JG, Pandey S, editors. Madison WI: ASA; 1999; pp. 69–81.

[16] Okogbenin E, Moreno I, Tomkins J, et al.: Marker-assisted breeding for cassava mosaic disease resistance. Translational genomics for crop breeding: biotic stress. 2013; 1: 291–325.

[17] Pujol BP, David P, McKey D: Microevolution in agricultural environments: How a traditional Amerindian farming practice favours heterozygosity in cassava (Manihot esculenta Crantz, Euphorbiaceae). Ecol. Lett. 2005; 8: 138–147.

[18] Rojas MC, Perez JC, Cellabos H, et al.: Analysis of inbreeding depression in eight S₁cassava families. Crop Sci. 2009; 49: 543–548.

[19] Scotti C, Pupilli F, Salvi S, et al.: Variation in vigor and in RFLP-estimated heterozygosity by selfing tetraploid alfalfa: new perspectives for the use of selfing in alfalfa breeding. Theor. Appl. Genetics. 2000; 101: 120–125.

[20] Vencovsky R, Barriga P: Genética Biométrica no Fitomelhoramento. Ribeirão Preto, Brazil: Sociedade Brasileira de Genética; 1992; p. 486.

Analysis of inbreeding depression in five S1 Cassava families of elite varieties at the seedling nursery and clonal evaluation trial stages.

Abstract

Background

Method

Results

Conclusion

Keywords

Introduction

Method

Study site

Population development

Single nucleotide polymorphisms (SNPs) markers genotyping

Data collections

Data analysis

Results

Origin of elite clones and size of their respective S1 families

Table 1. Origin of the elite clones and sizes of their respective S1 families evaluated in both trials.

Effects of inbreeding depression on pests and diseases

Table 2. Inbreeding depression measured in the five S1 cassava families based on the disease severity at the seedling nursery trial.

Table 3. Inbreeding depression measured in the five S1 cassava families based on the disease severity at the clonal evaluation trial.

Effects of inbreeding depression on agronomic traits

Table 4. Inbreeding depression measured in five S1 cassava (Manihot esculenta Crantz) families based on the yield traits in seedling nursery trial stage.

Table 5. Inbreeding depression measured in five S1 cassava (Manihot esculenta Crantz) families based on the yield traits in clonal evaluation trial stage.

Effects of inbreeding depression on morphological/architecture traits

Paired (dependent) sample t-test: Seedling nursery stage vs. Clonal evaluation stage

Table 6. Paired (dependent) samples t-test analysis: Seedling nursery trial vs. Clonal evaluation trial stages.

SNP estimates for heterozygosity

Figure 1. The effect of number of SNPs on the heterozygosity index with the exception of TMS 98/0002.

Table 7. Heterozygosity index measured in five S1 cassava (Manihot esculenta Crantz) families.

Figure 2. Distribution of S1 progenies (except TMS 98/0002) based on their percentage heterozygosity using SNPs markers.

Discussion

Conclusion

Data availability statement

Acknowledgments

References

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Analysis of inbreeding depression in five S₁ Cassava families of elite varieties at the seedling nursery and clonal evaluation trial stages.

Origin of elite clones and size of their respective S₁ families

Table 1. Origin of the elite clones and sizes of their respective S₁ families evaluated in both trials.

Table 2. Inbreeding depression measured in the five S₁ cassava families based on the disease severity at the seedling nursery trial.

Table 3. Inbreeding depression measured in the five S₁ cassava families based on the disease severity at the clonal evaluation trial.

Table 4. Inbreeding depression measured in five S₁ cassava (Manihot esculenta Crantz) families based on the yield traits in seedling nursery trial stage.

Table 5. Inbreeding depression measured in five S₁ cassava (Manihot esculenta Crantz) families based on the yield traits in clonal evaluation trial stage.

Table 7. Heterozygosity index measured in five S₁ cassava (Manihot esculenta Crantz) families.