Molecular Tools to Infer Resistance-Breaking Abilities of Rice Yellow Mottle Virus Isolates.

Rice yellow mottle virus (RYMV) is a major biotic constraint to rice cultivation in Africa. RYMV shows a high genetic diversity. Viral lineages were defined according to the coat protein (CP) phylogeny. Varietal selection is considered as the most efficient way to manage RYMV. Sources of high resistance were identified mostly in accessions of the African rice species, Oryza glaberrima. Emergence of resistance-breaking (RB) genotypes was observed in controlled conditions. The RB ability was highly contrasted, depending on the resistance sources and on the RYMV lineages. A molecular marker linked to the adaptation to susceptible and resistant O. glaberrima was identified in the viral protein genome-linked (VPg). By contrast, as no molecular method was available to identify the hypervirulent lineage able to overcome all known resistance sources, plant inoculation assays were still required. Here, we designed specific RT-PCR primers to infer the RB abilities of RYMV isolates without greenhouse experiments or sequencing steps. These primers were tested and validated on 52 isolates, representative of RYMV genetic diversity. The molecular tools described in this study will contribute to optimizing the deployment strategy of resistant lines, considering the RYMV lineages identified in fields and their potential adaptability.

Characterization of Blast Resistance in a Diverse Rice Panel from Sub-Saharan Africa.

There is a recent unparalleled increase in demand for rice in sub-Saharan Africa, yet its production is affected by blast disease. Characterization of blast resistance in adapted African rice cultivars can provide important information to guide growers and rice breeders. We used molecular markers for known blast resistance genes (Pi genes; n = 21) to group African rice genotypes (n = 240) into similarity clusters. We then used greenhouse-based assays to challenge representative rice genotypes (n = 56) with African isolates (n = 8) of Magnaporthe oryzae which varied in virulence and genetic lineage. The markers grouped rice cultivars into five blast resistance clusters (BRC) which differed in foliar disease severity. Using stepwise regression, we found that the Pi genes associated with reduced blast severity were Pi50 and Pi65, whereas Pik-p, Piz-t, and Pik were associated with increased susceptibility. All rice genotypes in the most resistant cluster, BRC 4, possessed Pi50 and Pi65, the only genes that were significantly associated with reduced foliar blast severity. Cultivar IRAT109, which contains Piz-t, was resistant against seven African M. oryzae isolates, whereas ARICA 17 was susceptible to eight isolates. The popular Basmati 217 and Basmati 370 were among the most susceptible genotypes. These findings indicate that most tested genes were not effective against African blast pathogen collections. Pyramiding genes in the Pi2/9 multifamily blast resistance cluster on chromosome 6 and Pi65 on chromosome 11 could confer broad-spectrum resistance capabilities. To gain further insights into genomic regions associated with blast resistance, gene mapping could be conducted with resident blast pathogen collections. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

The MYB transcription factor Seed Shattering 11 controls seed shattering by repressing lignin synthesis in African rice.

African cultivated rice (Oryza glaberrima Steud.) was domesticated from its wild progenitor species (Oryza barthii) about 3000 years ago. Seed shattering is one of the main constraints on grain production in African cultivated rice, which causes severe grain losses during harvest. By contrast, Asian cultivated rice (Oryza sativa) displays greater resistance to seed shattering, allowing higher grain production. A better understanding in regulation of seed shattering would help to improve harvesting efficiency in African cultivated rice. Here, we report the map-based cloning and characterization of OgSH11, a MYB transcription factor controlling seed shattering in O. glaberrima. OgSH11 represses the expression of lignin biosynthesis genes and lignin deposition by binding to the promoter of GH2. We successfully developed a new O. glaberrima material showing significantly reduced seed shattering by knockout of SH11 in O. glaberrima using CRISPR-Cas9 mediated approach. Identification of SH11 not only supplies a new target for seed shattering improvement in African cultivated rice, but also provides new insights into the molecular mechanism of abscission layer development.

State of ex situ conservation of landrace groups of 25 major crops.

Crop landraces have unique local agroecological and societal functions and offer important genetic resources for plant breeding. Recognition of the value of landrace diversity and concern about its erosion on farms have led to sustained efforts to establish ex situ collections worldwide. The degree to which these efforts have succeeded in conserving landraces has not been comprehensively assessed. Here we modelled the potential distributions of eco-geographically distinguishable groups of landraces of 25 cereal, pulse and starchy root/tuber/fruit crops within their geographic regions of diversity. We then analysed the extent to which these landrace groups are represented in genebank collections, using geographic and ecological coverage metrics as a proxy for genetic diversity. We find that ex situ conservation of landrace groups is currently moderately comprehensive on average, with substantial variation among crops; a mean of 63% ± 12.6% of distributions is currently represented in genebanks. Breadfruit, bananas and plantains, lentils, common beans, chickpeas, barley and bread wheat landrace groups are among the most fully represented, whereas the largest conservation gaps persist for pearl millet, yams, finger millet, groundnut, potatoes and peas. Geographic regions prioritized for further collection of landrace groups for ex situ conservation include South Asia, the Mediterranean and West Asia, Mesoamerica, sub-Saharan Africa, the Andean mountains of South America and Central to East Asia. With further progress to fill these gaps, a high degree of representation of landrace group diversity in genebanks is feasible globally, thus fulfilling international targets for their ex situ conservation.

The genetic control of glabrous glume during African rice domestication.

African cultivated rice, Oryza glaberrima, is characterized by its glabrous glumes. During domestication, the pubescent glumes of its wild ancestor, Oryza barthii, lost their trichomes, and in this study, we show that glabrous glume 5 (GLAG5), a WUSCHEL-like homeobox transcription factor gene on chromosome 5, is required for trichome development. DNA methylation associated with an hAT transposable element inserted in the promoter region of GLAG5 is found to reduce its expression, leading to the formation of glabrous glumes and leaves in African cultivated rice. Among 82 African cultivated rice varieties investigated in this study, 59 (approximately 71%) lines exhibit glabrous glumes and harbor the hAT transposon; however, the other 23 varieties (approximately 29%), which exhibit pubescent glumes, lack the hAT transposon, indicating that glag5 had undergone strong artificial selection. The πw/πc ratios also show the hAT transposon insertions influence the genetic diversity of an approximately 150-kb interval encompassing the GLAG5 locus. The identification of the GLAG5 gene provides new insights into the domestication of cultivated rice in Africa. We speculate that the selection of varieties with mutations in their promoter regions is an important aspect of crop domestication.

Development and validation of diagnostic SNP markers for quality control genotyping in a collection of four rice (Oryza) species.

Morphological identification of closely related rice species, particularly those in the Oryza AA genome group, presents major challenges and often results in cases of misidentification. Recent work by this group identified diagnostic single nucleotide polymorphic (SNP) markers specific for several rice species and subspecies based on DArTseq next-generation sequencing technology ("DArTseq"). These SNPs can be used for quality control (QC) analysis in rice breeding and germplasm maintenance programs. Here, we present the DArTseq-based diagnostic SNPs converted into Kompetitive allele-specific PCR (KASPar or KASP) assays and validation data for a subset of them; these can be used for low-cost routine genotyping quality control (QC) analysis. Of the 224 species/subspecies' diagnostic SNPs tested, 158 of them produced working KASP assays, a conversion success rate of 70%. Two validation experiments were run with 87 of the 158 SNP markers to ensure that the assays amplified, were polymorphic, and distinguished the five species/subspecies tested. Based on these validation test results, we recommend a panel of 36 SNP markers that clearly delineate O. barthii, O. glaberrima, O. longistaminata, O. sativa spp. indica and japonica. The KASP assays provide a flexible, rapid turnaround and cost-effective tool to facilitate germplasm curation and management of these four Oryza AA genome species across multiple genebanks.

Phytosanitary Interventions for Safe Global Germplasm Exchange and the Prevention of Transboundary Pest Spread: The Role of CGIAR Germplasm Health Units.

The inherent ability of seeds (orthodox, intermediate, and recalcitrant seeds and vegetative propagules) to serve as carriers of pests and pathogens (hereafter referred to as pests) and the risk of transboundary spread along with the seed movement present a high-risk factor for international germplasm distribution activities. Quarantine and phytosanitary procedures have been established by many countries around the world to minimize seed-borne pest spread by screening export and import consignments of germplasm. The effectiveness of these time-consuming and cost-intensive procedures depends on the knowledge of pest distribution, availability of diagnostic tools for seed health testing, qualified operators, procedures for inspection, and seed phytosanitation. This review describes a unique multidisciplinary approach used by the CGIAR Germplasm Health Units (GHUs) in ensuring phytosanitary protection for the safe conservation and global movement of germplasm from the 11 CGIAR genebanks and breeding programs that acquire and distribute germplasm to and from all parts of the world for agricultural research and food security. We also present the challenges, lessons learned, and recommendations stemming from the experience of GHUs, which collaborate with the national quarantine systems to export and distribute about 100,000 germplasm samples annually to partners located in about 90 to 100 countries. Furthermore, we describe how GHUs adjust their procedures to stay in alignment with evolving phytosanitary regulations and pest risk scenarios. In conclusion, we state the benefits of globally coordinated phytosanitary networks for the prevention of the intercontinental spread of pests that are transmissible through plant propagation materials.

Genetics and Genomics of African Rice (Oryza glaberrima Steud) Domestication.

African rice (Oryza glaberrima Steud) is one of the two independently domesticated rice species, the other one being Asian rice (Oryza sativa L.). Despite major progress being made in understanding the evolutionary and domestication history of African rice, key outstanding issues remain controversial. There appears to be an underlying difficulty in identifying the domestication centre and number of times the crop has been domesticated. Advances in genomics have provided unprecedented opportunities for understanding the genetic architecture of domestication related traits. For most of the domestication traits, the underlying genes and mutations have been identified. Comparative analysis of domestication genes between Asian and African rice has revealed that the two species went through an independent but convergent evolution process. The genetic and developmental basis of some of the domestic traits are conserved not only between Asian and African rice but also with other domesticated crop species. Analysis of genome data and its interpretation is emerging as a major challenge facing studies of domestication in African rice as key studies continue giving contradictory findings and conclusions. Insights obtained on the domestication of this species are vital for guiding crop improvement efforts.

Sequence Variants Linked to Key Traits in Interspecific Crosses between African and Asian Rice.

Asian and African rice gene pools vary in many traits that are important in rice breeding. The genetic basis of these differences was evaluated by analysis of important agronomic traits in crosses between African and Asian rice. Trait-associated variants (TAVs) influencing three quantitative agronomic traits, heading date (Hd), tiller number at maturity (T), and 1000 grain weight (TGW), were identified by association analysis of crosses between Asian and African rice. Populations were developed by crossing WAB56-104 (Oryza sativa) and CG14 (Oryza glaberrima). DNA from plants with extremely high or low values for these phenotypes was bulked and sequenced. The reference genome of O. sativa cv Nipponbare was used in general association analysis and candidate gene analysis. A total of 5152 non-synonymous single nucleotide polymorphisms (SNPs) across 3564 genes distinguished the low and the high bulks for Hd, T, and TGW traits; 611 non-synonymous SNPs across 447 genes were found in KEGG pathways. Six non-synonymous SNPs were found in the sequences of LOC107275952, LOC4334529, LOC4326177, LOC107275432, LOC4335790, and LOC107275425 genes associated with Hd, T, and TGW traits. These genes were involved in: abscisic-acid biosynthesis, carotenoid biosynthesis, starch and sucrose metabolism, and cytokinin biosynthesis. Analysis of 24 candidate genes associated with Hd, T, and TGW traits showed seven non-synonymous variations in the sequence of Hd3a and Ehd2 from the Hd genes (not in a KEGG pathway), D10 and D53 from the T genes (strigolactones biosynthetic pathway), and Gn1a and GIF1 from the TGW genes (cytokinin biosynthetic and starch and sucrose metabolism pathways). This study identified significant differences in allele frequencies supported by high sequence depth in analysis of bulks displaying high and low values for these key traits. These trait-associated variants are likely to be useful in rice improvement.

Germplasm Acquisition and Distribution by CGIAR Genebanks.

The international collections of plant genetic resources for food and agriculture (PGRFA) hosted by 11 CGIAR Centers are important components of the United Nations Food and Agriculture Organization's global system of conservation and use of PGRFA. They also play an important supportive role in realizing Target 2.5 of the Sustainable Development Goals. This paper analyzes CGIAR genebanks' trends in acquiring and distributing PGRFA over the last 35 years, with a particular focus on the last decade. The paper highlights a number of factors influencing the Centers' acquisition of new PGRFA to include in the international collections, including increased capacity to analyze gaps in those collections and precisely target new collecting missions, availability of financial resources, and the state of international and national access and benefit-sharing laws and phytosanitary regulations. Factors contributing to Centers' distributions of PGRFA included the extent of accession-level information, users' capacity to identify the materials they want, and policies. The genebanks' rates of both acquisition and distribution increased over the last decade. The paper ends on a cautionary note concerning the potential of unresolved tensions regarding access and benefit sharing and digital genomic sequence information to undermine international cooperation to conserve and use PGRFA.

Comparisons of sampling methods for assessing intra- and inter-accession genetic diversity in three rice species using genotyping by sequencing.

To minimize the cost of sample preparation and genotyping, most genebank genomics studies in self-pollinating species are conducted on a single individual to represent an accession, which may be heterogeneous with larger than expected intra-accession genetic variation. Here, we compared various population genetics parameters among six DNA (leaf) sampling methods on 90 accessions representing a wild species (O. barthii), cultivated and landraces (O. glaberrima, O. sativa), and improved varieties derived through interspecific hybridizations. A total of 1,527 DNA samples were genotyped with 46,818 polymorphic single nucleotide polymorphisms (SNPs) using DArTseq. Various statistical analyses were performed on eleven datasets corresponding to 5 plants per accession individually and in a bulk (two sets), 10 plants individually and in a bulk (two sets), all 15 plants individually (one set), and a randomly sampled individual repeated six times (six sets). Overall, we arrived at broadly similar conclusions across 11 datasets in terms of SNP polymorphism, heterozygosity/heterogeneity, diversity indices, concordance among genetic dissimilarity matrices, population structure, and genetic differentiation; there were, however, a few discrepancies between some pairs of datasets. Detailed results of each sampling method, the concordance in their outputs, and the technical and cost implications of each method were discussed.

Advances in Molecular Genetics and Genomics of African Rice (Oryza glaberrima Steud).

African rice (Oryza glaberrima) has a pool of genes for resistance to diverse biotic and abiotic stresses, making it an important genetic resource for rice improvement. African rice has potential for breeding for climate resilience and adapting rice cultivation to climate change. Over the last decade, there have been tremendous technological and analytical advances in genomics that have dramatically altered the landscape of rice research. Here we review the remarkable advances in knowledge that have been witnessed in the last few years in the area of genetics and genomics of African rice. Advances in cheap DNA sequencing technologies have fuelled development of numerous genomic and transcriptomic resources. Genomics has been pivotal in elucidating the genetic architecture of important traits thereby providing a basis for unlocking important trait variation. Whole genome re-sequencing studies have provided great insights on the domestication process, though key studies continue giving conflicting conclusions and theories. However, the genomic resources of African rice appear to be under-utilized as there seems to be little evidence that these vast resources are being productively exploited for example in practical rice improvement programmes. Challenges in deploying African rice genetic resources in rice improvement and the genomics efforts made in addressing them are highlighted.

Comparisons of molecular diversity indices, selective sweeps and population structure of African rice with its wild progenitor and Asian rice.

KEY MESSAGE: The extent of molecular diversity parameters across three rice species was compared using large germplasm collection genotyped with genomewide SNPs and SNPs that fell within selective sweep regions. Previous studies conducted on limited number of accessions have reported very low genetic variation in African rice (Oryza glaberrima Steud.) as compared to its wild progenitor (O. barthii A. Chev.) and to Asian rice (O. sativa L.). Here, we characterized a large collection of African rice and compared its molecular diversity indices and population structure with the two other species using genomewide single nucleotide polymorphisms (SNPs) and SNPs that mapped within selective sweeps. A total of 3245 samples representing African rice (2358), Asian rice (772) and O. barthii (115) were genotyped with 26,073 physically mapped SNPs. Using all SNPs, the level of marker polymorphism, average genetic distance and nucleotide diversity in African rice accounted for 59.1%, 63.2% and 37.1% of that of O. barthii, respectively. SNP polymorphism and overall nucleotide diversity of the African rice accounted for 20.1-32.1 and 16.3-37.3% of that of the Asian rice, respectively. We identified 780 SNPs that fell within 37 candidate selective sweeps in African rice, which were distributed across all 12 rice chromosomes. Nucleotide diversity of the African rice estimated from the 780 SNPs was 8.3 × 10-4, which is not only 20-fold smaller than the value estimated from all genomewide SNPs (π = 1.6 × 10-2), but also accounted for just 4.1%, 0.9% and 2.1% of that of O. barthii, lowland Asian rice and upland Asian rice, respectively. The genotype data generated for a large collection of rice accessions conserved at the AfricaRice genebank will be highly useful for the global rice community and promote germplasm use.

Development of species diagnostic SNP markers for quality control genotyping in four rice (Oryza L.) species.

Species misclassification (misidentification) and handling errors have been frequently reported in various plant species conserved at diverse gene banks, which could restrict use of germplasm for correct purpose. The objectives of the present study were to (i) determine the extent of genotyping error (reproducibility) on DArTseq-based single-nucleotide polymorphisms (SNPs); (ii) determine the proportion of misclassified accessions across 3134 samples representing three African rice species complex (Oryza glaberrima, O. barthii, and O. longistaminata) and an Asian rice (O. sativa), which are conserved at the AfricaRice gene bank; and (iii) develop species- and sub-species (ecotype)-specific diagnostic SNP markers for rapid and low-cost quality control (QC) analysis. Genotyping error estimated from 15 accessions, each replicated from 2 to 16 times, varied from 0.2 to 3.1%, with an overall average of 0.8%. Using a total of 3134 accessions genotyped with 31,739 SNPs, the proportion of misclassified samples was 3.1% (97 of the 3134 accessions). Excluding the 97 misclassified accessions, we identified a total of 332 diagnostic SNPs that clearly discriminated the three indigenous African species complex from Asian rice (156 SNPs), O. longistaminata accessions from both O. barthii and O. glaberrima (131 SNPs), and O. sativa spp. indica from O. sativa spp. japonica (45 SNPs). Using chromosomal position, minor allele frequency, and polymorphic information content as selection criteria, we recommended a subset of 24 to 36 of the 332 diagnostic SNPs for routine QC genotyping, which would be highly useful in determining the genetic identity of each species and correct human errors during routine gene bank operations.

The Rise and Fall of African Rice Cultivation Revealed by Analysis of 246 New Genomes.

African rice (Oryza glaberrima) was domesticated independently from Asian rice. The geographical origin of its domestication remains elusive. Using 246 new whole-genome sequences, we inferred the cradle of its domestication to be in the Inner Niger Delta. Domestication was preceded by a sharp decline of most wild populations that started more than 10,000 years ago. The wild population collapse occurred during the drying of the Sahara. This finding supports the hypothesis that depletion of wild resources in the Sahara triggered African rice domestication. African rice cultivation strongly expanded 2,000 years ago. During the last 5 centuries, a sharp decline of its cultivation coincided with the introduction of Asian rice in Africa. A gene, PROG1, associated with an erect plant architecture phenotype, showed convergent selection in two rice cultivated species, Oryza glaberrima from Africa and Oryza sativa from Asia. In contrast, a shattering gene, SH5, showed selection signature during African rice domestication, but not during Asian rice domestication. Overall, our genomic data revealed a complex history of African rice domestication influenced by important climatic changes in the Saharan area, by the expansion of African agricultural society, and by recent replacement by another domesticated species.

Genetic control of seed shattering during African rice domestication.

Domestication represents a unique opportunity to study the evolutionary process. The elimination of seed dispersal traits was a key step in the evolution of cereal crops under domestication. Here, we show that ObSH3, a YABBY transcription factor, is required for the development of the seed abscission layer. Moreover, selecting a genomic segment deletion containing SH3 resulted in the loss of seed dispersal in populations of African cultivated rice (Oryza glaberrima Steud.). Functional characterization of SH3 and SH4 (another gene controlling seed shattering on chromosome 4) revealed that multiple genes can lead to a spectrum of non-shattering phenotypes, affecting other traits such as ease of threshing that may be important to tune across different agroecologies and postharvest practices. The molecular evolution analyses of SH3 and SH4 in a panel of 93 landraces provided unprecedented geographical detail of the domestication history of African rice, tracing multiple dispersals from a core heartland and introgression from local wild rice. The cloning of ObSH3 not only provides new insights into a critical crop domestication process but also adds to the body of knowledge on the molecular mechanism of seed dispersal.

Assessment of Genetic Variation and Population Structure of Diverse Rice Genotypes Adapted to Lowland and Upland Ecologies in Africa Using SNPs.

Using interspecific crosses involving Oryza glaberrima Steud. as donor and O. sativa L. as recurrent parents, rice breeders at the Africa Rice Center developed several 'New Rice for Africa (NERICA)' improved varieties. A smaller number of interspecific and intraspecific varieties have also been released as 'Advanced Rice for Africa (ARICA)'. The objective of the present study was to investigate the genetic variation, relatedness, and population structure of 330 widely used rice genotypes in Africa using DArTseq-based single nucleotide polymorphisms (SNPs). A sample of 11 ARICAs, 85 NERICAs, 62 O. sativa spp. japonica, and 172 O. sativa spp. indica genotypes were genotyped with 27,560 SNPs using diversity array technology (DArT)-based sequencing (DArTseq) platform. Nearly 66% of the SNPs were polymorphic, of which 15,020 SNPs were mapped to the 12 rice chromosomes. Genetic distance between pairs of genotypes that belong to indica, japonica, ARICA, and NERICA varied from 0.016 to 0.623, from 0.020 to 0.692, from 0.075 to 0.763, and from 0.014 to 0.644, respectively. The proportion of pairs of genotypes with genetic distance > 0.400 was the largest within NERICAs (35.1% of the pairs) followed by ARICAs (18.2%), japonica (17.4%), and indica (5.6%). We found one pair of japonica, 11 pairs of indica, and 35 pairs of NERICA genotypes differing by <2% of the total scored alleles, which was due to 26 pairs of genotypes with identical pedigrees. Cluster analysis, principal component analysis, and the model-based population structure analysis all revealed two distinct groups corresponding to the lowland (primarily indica and lowland NERICAs) and upland (japonica and upland NERICAs) growing ecologies. Most of the interspecific lowland NERICAs formed a sub-group, likely caused by differences in the O. glaberrima genome as compared with the indica genotypes. Analysis of molecular variance revealed very great genetic differentiation (FST = 0.688) between the lowland and upland ecologies, and 31.2% of variation attributable to differences within cluster groups. About 8% (1,197 of 15,020) of the 15,020 SNPs were significantly (P < 0.05) different between the lowland and upland ecologies and formed contrasting haplotypes that could clearly discriminate lowland from upland genotypes. This is the first study using high density markers that characterized NERICA and ARICA varieties in comparison with indica and japonica varieties widely used in Africa, which could aid rice breeders on parent selection for developing new improved rice germplasm.

Role of genomics in promoting the utilization of plant genetic resources in genebanks.

Global efforts have seen the world's plant genetic resources (PGRs) conserved in about 1625 germ plasm repositories. Utility of these resources is important in increasing the resilience and productivity of agricultural production systems. However, despite their importance, utility of these resources has been poor. This article reviews the real and potential application of the current advances in genomic technologies in improving the utilization of these resources. The actual and potential application of these genomic approaches in plant identification, phylogenetic analysis, analysing the genetic value of germ plasm, facilitating germ plasm selection in genebanks as well as instilling confidence in international germ plasm exchange system is discussed. We note that if genebanks are to benefit from this genomic revolution, there is need for fundamental changes in the way genebanks are managed, perceived, organized and funded. Increased collaboration between genebank managers and the user community is also recommended.

Sequencing of bulks of segregants allows dissection of genetic control of amylose content in rice.

Amylose content (AC) is a key quality trait in rice. A cross between Oryza glaberrima (African rice) and Oryza sativa (Asian rice) segregating for AC was analysed by sequencing bulks of individuals with high and low AC. SNP associated with the granule bound starch synthase (GBSS1) locus on chromosome 6 were polymorphic between the bulks. In particular, a G/A SNP that would result in an Asp to Asn mutation was identified. This amino acid substitution may be responsible for differences in GBSS activity as it is adjacent to a disulphide linkage conserved in all grass GBSS proteins. Other polymorphisms in genomic regions closely surrounding this variation may be the result of linkage drag. In addition to the variant in the starch biosynthesis gene, SNP on chromosomes 1 and 11 linked to AC was also identified. SNP was found in the genes encoding the NAC and CCAAT-HAP5 transcription factors that have previously been linked to starch biosynthesis. This study has demonstrated that the approach of sequencing bulks was able to identify genes on different chromosomes associated with this complex trait.