African Rice Research Articles

Impact of Rice Stem Borers and Identification of <i>Orseolia oryzivora</i> Harris & Gagné, 1982 (Diptera: Cecidomyiidae) Biotypes in the Southern Cameroon

The study compares the impact due to rice stem borers in two sites (Yaounde and Ntui). It also shows the diversity of the African Rice Gall Midge (AfRGM) biotypes in southern Cameroon (Santchou, Ndop, Tonga, Ebolowa, Ba?gom, Yaounde and Ntui). The New Rice for Africa (NERICA) varieties 3, 8, 9 and 13 sown in Ntui were less attacked than those sown in Yaounde. At both sites, damages ranged from 0.78% to 2.7%. In terms of diversity, the main stem-borer species were O. oryzivora, Diopsis apicalis, D. longiconis and Chilo zacconius. Molecular analyses of Orseolia oryzivora larvae collected in the localities of Santchou, Ndop, Tonga, Ebolowa, Ba?gom and Yaounde showed the existence of more than one O. oryzivora biotype in southern Cameroon’s rice basins.

Moving toward Rice Self-Sufficiency in Sub-Saharan Africa by 2030: Lessons Learnt from 10 Years of the Coalition for African Rice Development

The Coalition for African Rice Development (CARD) was initiated as policy framework with the aim of doubling rice production in sub-Saharan Africa (SSA) over the period from 2008 to 2018. This paper assesses the contribution of the CARD policy to rice production and forecasts the local rice supply and demand for a better understanding of the policies needed to attain rice self-sufficiency by 2030. A combination of the autoregressive integrated moving average method and counterfactual approach was adopted using rice statistical data from 23 countries in SSA. Results showed that the contribution of CARD to paddy rice production in 2018 was 10.2 million ton, equivalent to 74% of target. This resulted from the increasing of area and yield by 23% and 19%, respectively. However, yield growth rate was not sustainable in almost two-third of countries. Investments on supply-push factors such as fertilizer and irrigation development, which was the focus in the past, have less effect on the rice production. We conclude that sustainable investments on demand-pull factors such as private-led modern milling sector and contract farming development should be prioritized for achieving rice self-sufficiency in SSA.

Multi-elemental analysis of roots and leaves from Oryza glaberrima rice plants at vegetative stage of growth by combined PIGE, RBS, PIXE and GC-TDS methods

This study aims at understanding the bioaccumulation and transport mechanisms of both micronutrients and macronutrients in the leaves and roots of African rice Oryza glaberrima Steud (OG) plants cultivated in Senegal during the wet season at the vegetative stage of growth and to evaluate the suitability of OG rice plants for phytoremediation. A comparative analysis of the elemental composition of roots and leaves of OG plants, provided information on the bioaccumulation of nutrients necessary for plant growth which can become detrimental if their toxicity level is reached. The elemental analysis of basic nutrients (C, H, O), macronutrients (N, P, K, Ca, Mg and S) and micronutrients (Cl, Fe, Cu, Mn, Ni and Zn) in the OG roots and leaves was made possible by several elemental analytical techniques (PIGE, RBS, PIXE and GC-TDS). All methods were validated by analysis of pure substances and certified reference materials. The high accumulation rate of Cu (5 x), Al (4 x), Zr (≥ 2 x) and Fe (~ 2 x) in the OG rice roots relative to the leaves indicates the existence of low translocation factors for these metals from root to leaves probably due to the existence of plant mechanisms to limit their transport and to preferentially accumulate Si (5.2 ± 0.5 % mass) in the rice leaves. The high accumulation rates of Cr, Mn, Ni and Rb in the OG leaves relative to the roots, and of Al, Fe, Cu and Zr in the OG roots relative to the leaves, shows that these metals can be partially removed from soil through phytoextraction processes by harvesting the shoot and root tissues.

Morpho-Physiological Response of Oryza glaberrima to Gradual Soil Drying

Soil drought occurrence during dry season has been the main constraint, besides prolonged flooding during rainy season, in increasing cropping intensity and rice productivity in tropical riparian wetland. Use of drought tolerant rice genotype might be a suitable option for overcoming such problem. This study focused on the effects of gradual soil drying during early vegetative growth stage on morphological and physiological traits of five Oryza glaberrima genotypes, namely RAM12, RAM14, RAM59, RAM97 and RAM101, and two Oryza sativa subsp japonica genotypes, i.e. Koshihikari and Minamihatamochi. The plants were subjected to 6 d of gradual soil drying condition from 15 days after transplanting (DAT) to 20 DAT, and were allowed to recover until 22 DAT. Gradual soil drying reduced plant growth as indicated by dry mass accumulation. Drought reduced stomatal conductance and increased leaf rolling score of all the genotypes. All the genotypes showed comparable response on stomatal conductance, but O. glaberrima genotypes performed higher in leaf rolling recovery. Meanwhile, O. sativa genotypes decreased total leaf area and specific leaf area, but increased specific leaf weight in order to avoid further damages due to drought stress. Drought tolerance mechanisms in RAM101, RAM12, RAM59 and RAM14 were associated with leaf morpho-physiological responses, root traits and dry biomass accumulation.

High-resolution genetic mapping of a novel bacterial blight resistance gene xa-45(t) identified from Oryza glaberrima and transferred to Oryza sativa.

A novel recessive bacterial blight resistance locus designated as a xa-45(t) was identified from Oryza glaberrima accession IRGC 102600B, transferred to O. sativa and mapped to the long arm of chromosome 8 using ddRAD sequencing approach. The identified QTL spans 80kb region on Nipponbare reference genome IRGSP-1.0 and contains 9 candidate genes. An STS marker developed from the locusLOC_Os08g42410was found co-segregating with the trait and will be useful for marker-assisted transfer of this recessive resistance gene in breeding programs. Bacterial blight, caused by Xanthomonas oryzae pv. oryzae, is one of the major constraints of rice productivity in Southeast Asia. In spite of having 44 bacterial blight resistance genes from cultivated rice and wild species, the durability of resistance is always at stake due to the continually evolving nature of the pathogen and lack of suitable chemical control. Here, we report high-resolution genetic mapping of a novel bacterial blight resistance gene tentatively designated as a xa-45(t) from an introgression line derived from Oryzaglaberrimaaccession IRGC 102600B. This introgression line was crossed with the susceptible riceindicacultivar cv. Pusa 44 to generate F2and F2:3populations for inheritance and mapping studies. The inheritance studies revealed the presence of single recessive locus controlling resistance to the Xanthomonas pathotype seven. A high-density linkage map was constructed using double-digest restriction-associated DNA sequencing of 96 F2 populations along with the parents. The QTL mapping identified a major locus on the long arm of rice chromosome 8 with a LOD score of 33.22 between the SNP markers C8.26737175 and C8.26818765. The peak marker, C8.26810477, explains 49.8% of the total phenotypic variance and was positioned at 202.90cM on the linkage map. This major locus spans 80kb region on Nipponbare reference genome IRGSP-1.0 and contains 9 candidate genes. A co-segregating STS marker was developed from the LOC_Os08g42410 for efficient transfer of this novel gene to elite cultivars.

Physicochemical and manufacturing cost elements of complementary food formulations from broken fraction of rice cultivars, soybean and sorghum malt

The broken fractions of rice cultivars improved for yield, grain quality, amylose content and tolerance to common production constraints consisting of FARO 44, FARO 52, NERICA L-34, NERICA L-19 and LOCAL RICE. The rice cultivars, Sorghum malt and soybean were processed. Evaluation of the physicochemical properties of these new African rice cultivars together with their products yield and utilization (value addition) were the main objective of this study. The experimental design for these infant food formulations (5x2x2 factorial design) consisted of those 5 rice cultivars, sorghum malt (0, 5%) and soybeans flour (0, 30%). These formulations together with the raw materials were evaluated for their physicochemical properties. Manufacturing cost of formulations with ratios of rice to soybeans 70:30 with malt were selected and quantified. Results of particle size distributions showed significant (p < 0.05) difference. However, 98.38 and 94.90% of raw materials and blends respectively, successfully passed through 600 µm sieve aperture. Mesh 300 and 180 µm were found to retain the highest percentage particles. Functional properties of raw materials and blends were within the recommended range. More importantly, pH values of blends were comparable to infants’ natural milk drinks. Proximate composition of raw materials and blends contain 4.14 to 9.59% moisture, 0.37 to 5.12% ash, 8.25 to 45.35% protein, 0.41 to 20.00% fat and 26.54 to 82.59% carbohydrate. Blends fiber and energy ranged from 1.27 to 4.33 and 362.95 to 388.71 Kcal (1541.92 to 1643.04 KJ) respectively. Formulations with ratios of rice to soybeans 70:30 with malt and those of rice 100% alone with malt were observed suitable as follow-on formulae and for infant(s) with critical protein related ailments, respectively. Net profit generated is more significant than interest rate that could have been obtained from bank saving system

Multivariate analysis and selection criteria for identification of African rice(Oryza glaberrima)for genetic improvement ofindicarice cultivars

AbstractThirty-one accessions ofOryza glaberrimawere evaluated to study the genetic variability, correlation, path, principal component analysis (PCA) andD2analysis. Box plots depicted high estimates of variability for days to 50% flowering and grain yield per plant inKharif2016, plant height, productive tillers, panicle length and 1000 seed weight inKharif2017. Correlation studies revealed days to 50% flowering, plant height, panicle length, number of productive tillers, spikelets per panicle having a high direct positive association with grain yield, while path analysis identified the number of productive tillers having the maximum direct positive effect on grain yield. Days to 50% floweringviaspikelets per panicle, productive tillers and plant heightviaspikelets per panicle exhibited high positive indirect effects on grain yield per plant. PCA showed that a cumulative variance of 54.752% from yield per plant, days to 50% flowering, spikelets per panicle and panicle length, contributing almost all the variation of traits whileD2analysis identified days to 50% flowering and grain yield per plant contributing maximum to the genetic diversity. Therefore, selection of accessions with more number of productive tillers and early maturity would be most suitable for yield improvement programme. The study has revealed the utility of African rice germplasm and its potential to utilize in the genetic improvement ofindica rice varieties.

Two SNP Mutations Turned off Seed Shattering in Rice.

Seed shattering is an important agronomic trait in rice domestication. In this study, using a near-isogenic line (NIL-hs1) from Oryza barthii, we found a hybrid seed shattering phenomenon between the NIL-hs1 and its recurrent parent, a japonica variety Yundao 1. The heterozygotes at hybrid shattering 1 (HS1) exhibited the shattering phenotype, whereas the homozygotes from both parents conferred the non-shattering. The causal HS1 gene for hybrid shattering was located in the region between SSR marker RM17604 and RM8220 on chromosome 4. Sequence verification indicated that HS1 was identical to SH4, and HS1 controlled the hybrid shattering due to harboring the ancestral haplotype, the G allele at G237T site and C allele at C760T site from each parent. Comparative analysis at SH4 showed that all the accessions containing ancestral haplotype, including 78 wild relatives of rice and 8 African cultivated rice, had the shattering phenotype, whereas all the accessions with either of the homozygous domestic haplotypes at one of the two sites, including 17 wild relatives of rice, 111 African cultivated rice and 65 Asian cultivated rice, showed the non-shattering phenotype. Dominant complementation of the G allele at G237T site and the C allele at C760T site in HS1 led to a hybrid shattering phenotype. These results help to shed light on the nature of seed shattering in rice during domestication and improve the moderate shattering varieties adapted to mechanized harvest.

Ecology of the African Rice Gall Midge, <i>Orseolia oryzivora</i> (Diptera: Cecidomyiidae) in Western Burkina Faso, West Africa

The African rice midge, Orseolia oryzivora H. & G., is an important insect pest in Burkina Faso. Our study was conducted from 2017 to 2019 in three irrigated rice schemes including Banzon, Karfiguéla and Vallée du Kou in Western Burkina Faso. The objective of this study was to investigate the ecology of the insect pest. In each rice scheme, four plots of 500 m2 each, spread 50 m apart were randomly selected in farmers’ fields and used for the study. In each plot, a light trap was implemented along one of the diagonals of the plot. Each light trap consisted of a yellow plastic round bowl containing soapy water almost at half the bowl and a torch. The bowl had a capacity of 4.5 l, a diameter of 28 cm and a height of 10.5 cm. The torch had 3 batteries of 1.5 V each, 30 cm long and 1 cm diameter. The torches were lit every night at 6 pm and off at 6 am. Four hundred tillers were collected per month from the cultivated rice and from each of the alternative hosts of the rice midge in order to evaluate the monthly gall count, pre-imaginal populations and parasitism associated with O. oryzivora and its cousin, O. bonzii that is hosted by Paspalum scrobiculatum. The number of O. oryzivora’s adults caught in the light traps was higher during the wet season than the dry season. The level of larval parasitism ranged from 6.16% (Banzon) to 14.29% (Vallée du Kou). The highest level of pupal parasitism (13.73%) was recorded in Vallée du Kou. The damage of O. oryzivora was observed on the wild rice species, O. longistaminata in all three sites. The highest levels of parasitism associated with O. bonzii were recorded in Karfiguéla (20.08% and 25.21% for larval parasitism and pupal parasitism, respectively). These findings are useful in the development of a sustainable control strategy of O. oryzivora.

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.

Hidden Rice Diversity in the Guianas.

Traditional crop varieties are an important source of genetic diversity for crop adaptation and modern breeding. Landraces of Asian (Oryza sativa) and African (Oryza glaberrima) rice have been well studied on the continents where they were domesticated. However, their history of cultivation in northern South America is poorly understood. Here, we reveal the rice diversity that is maintained by Maroons, descendants of enslaved Africans who fled to the interior forests of the Guianas ca. 300 years ago. We interviewed subsistence farmers who practice shifting cultivation along the Maroni and Lawa rivers that form the natural border between French Guiana and Suriname, and used ethnobotanical and morphological methods to identify around 50 varieties, of which 15 were previously undocumented. The genetic origin of these varieties was explored using the Angiosperms353 universal probe set. Despite the large distances between sites and relative inaccessibility of the area, phenotypic and genetic diversity did not display any geographic structure, which is consistent with knowledge of seed exchange among members of the same ethnolinguistic group. Although improved US cultivars were introduced in Maroon villages in the 1940s, these have not displaced the traditional landraces, which are cherished for their taste and nutritious qualities and for their importance in Maroon spiritual life. The unique agricultural and ritual practices of Maroons confirm their role as custodians of rice diversity, a role that is currently under threat from external pressures and encroaching globalization. We expect that the rice diversity uncovered in this study represents only a fraction of the total diversity in the Guianas and may constitute a large untapped resource that holds promise for future rice improvement. Further efforts to inventory and preserve these landraces will help to protect a precious cultural heritage and local food security.

Genetic diversity and differentiation of the African wild rice (Oryza longistaminata chev. et roehr) in Ethiopia

Abstract Undomesticated rice species (Oryza longistaminata) which possessed AA genome as O. sativa, is native to Africa and a good candidate for widening the cultivated rice gene pool. So as to use O. longistaminata as a genetic resource in rice breeding, its level of genetic diversity and differentiation must be resolved. Thus, 57 SSR markers across the whole rice genome were used to assess the genetic diversity of 360 O. longistaminata accessions from 12 agroecologies of the Amhara and Gambella regions of Ethiopia. RADSeq based analysis on 87 early maturing O. longistaminata accessions was also done to assess genetic differentiation among the Amhara and Gambella regions of Ethiopia. In this study, the Ethiopian O. longistaminata populations showed high genetic diversity (PIC = 76.5%) with a region based genetic differentiation (Fst = 0.08). Furthermore, genetic relatedness among populations of a region was mainly laid on their geographic provenance. Hence, the high genetic diversity and region based differentiation observed in the Ethiopian O. longistaminata populations could be used as a potential genetic resource in rice improvement programs.

Genetic diversity and population structure analysis of Asian and African aromatic rice (Oryza sativa L.) genotypes

Rice germplasms collected from different regions could be used as valuable resources for the future breeding programme. For the utilization of such collections, knowledge about the level and distribution of genetic diversity among these collections will facilitate the breeder. In this study, we report the phenotypic correlation, biochemical quality parameters and population genetic analysis of 35 rice accessions including 34 aromatic rice from different countries and a nonaromatic, Nagina 22, a well-known drought resistance variety. Further biochemical quality analysis, gel consistency test, molecular diversity analysis with 55 simple sequence repeat markers, population structure analysis and pair wise FST analysis were also conducted to assess the genetic diversity. The collected rice genotypes showed significant variability in different agronomic traits, i.e. spikelet per panicle, branches per panicle etc. Results obtained from the above tests demonstrated the importance of regional genetic studies for understanding the diversification of aromatic rice in Asian and African rice.

Determination of Arsenic Content in Different Brands of Rice Sold in Port Harcourt, Nigeria

Arsenic, a naturally occurring element seen in the environment and cycled by water has been seen to be a pollutant in soil, water and air. The inorganic form of arsenic is associated with major health problems and cancer mainly arising due to its anthropogenic activities. The content of arsenic was assayed in six rice brands sold in Port Harcourt, Rivers State, Nigeria. The rice samples were ground to powdered forms and subjected to acid digestion before analysis. The Agilent Micro Plasma Atomic Emission Spectrophotometer (MP-AES) was used to analyze the digested samples. Results showed mean arsenic concentration in Vico Rice, the highest as 1.0073. This was followed by Local Rice 0.9420, Marori Benz 0.9140, Golden Stallion Rice 0.861 and the Cap Rice 0.8077 while the least mean concentration of arsenic was recorded in African Princess Rice 0.6417, all in mg/kg. The mean concentration was significantly different in the six brands of rice (P<0.05). The arsenic content in the different rice brand was within the tolerable concentration of 1.4 mg/kg as in cereals and vegetables. This concentration did not exceed the Maximum Permissible Limit of 1 mg/kg hence considered safe for consumption except Vico rice with a higher concentration above 1 mg/kg. This shows that rice brands sold in Port Harcourt pose no health risk for consumers with respect to arsenic content.

English

This study aims to analyse the factors that influence the selection of governance structures by rice producers and processors in Benin. Unlike previous studies, the factors that influence the selection of governance structures are identified simultaneously for both producers and processors of paddy. Data were collected in Benin from about 300 producers and 140 processors of paddy randomly selected. The results indicate that 78% of producers and 92% of the processors use spot market for paddy transaction. Around a quarter of producers use at least two governance structures to sell paddy. Producers and processors belonging to an innovation platform are more likely to use formal contracts in their transactions. Also, producers and processors are more likely to use credit payment mechanism with formal contract. The findings suggest that innovation platforms can be used to facilitate contractual relationships between paddy producers and processors. Key words: Multivariate probit, market dynamics, African rice value chains, governance mechanisms. &nbsp

NMR chemical shift backbone assignment of the viral protein P1 encoded by the African Rice Yellow Mottle Virus.

RNA silencing describes a pan-eukaryotic pathway of gene regulation where doubled stranded RNA are processed by the RNAse III enzyme Dicer or homologs. In particular, plants use it as a way to defend themselves against pathogen invasions. In turn, to evade the plant immune response, viruses have developed anti-RNA silencing mechanisms. They may indeed code for proteins called "viral suppressor of RNA silencing" which block the degrading of viral genomic or messenger RNA by the plant. The Rice Mottle Virus is an African virus of the sobemovirus family, which attacks the most productive rice varieties cultivated on this continent. It encodes P1, a cysteine-rich protein described as a potential RNA silencing suppressor. P1 is a 157 amino-acid long protein, characterized by a high propensity to aggregate concomitant with a limited stability with time in the conditions used in structural studies. To overcome this problem, shorter fragments were also studied. This strategy enabled the assignment of more than 90% backbone resonances of P1. This assignment should set the base of future NMR investigation of the protein structure and of its interactions with rice cellular partners.

Understanding and breaking down the reproductive barrier between Asian and African cultivated rice: a new start for hybrid rice breeding.

Understanding and breaking down the reproductive barrier between Asian and African cultivated rice: a new start for hybrid rice breeding.

Rice husk and saw dust as filter loss control agents for water-based muds

When drilling with water based muds (WBM), significant fluid loss volumes from the mud into the formation can have adverse effects not just on the mud and its properties but also on the stability of the wellbore. Prevention of mud filter loss is one way of assessing the performance of a drilling mud. However, evaluation of the effectiveness or otherwise of a fluid loss control additive can be made by characterizing the mud cake formed. Interestingly, the mud cake characterization is one area that has been somewhat neglected in drilling fluid formulation with agro waste materials. Two cellulosic materials - rice husk and saw dust were chosen for the experimental study. The specie of the rice husk used was the African rice (Oryza glaberrima) while the dust from the saw milling of Oxystigma manni was utilized for this study. To ensure result acceptability, the rice husk and saw dust were ground and the resulting products were sieved to 1.25 × 10−4 m. The filtration characteristics of the formulated mud samples were tested using the American Petroleum Institute (API) filter press and in accordance to the API recommended practice for field testing WBMs. From the filter loss tests, it was observed that the ground rice husk prevented filter loss by an average of 77% compared to ground saw dust filtration control of 63%. In addition, it was observed that at higher concentrations, ground saw dust and rice husk prevented fluid loss to the minimum acceptable API standard. For the filter cake thickness measured in millimetres, ground rice husk exhibited thicker mud cakes when compared with the saw dust by an average amount of 14%. For the mud cake characteristics, the rice husk mud exhibited smooth and slippery cakes while the saw dust mud exhibited rough texture, sticky and firm cakes.

Effect of Beauveria bassiana and Metarhizium anisopliae on the adult African rice gall midge (AfRGM – Orseolia oryzivora)

Rice is an important staple crop whose production is limited by array of insect pests and diseases. African rice gall midge (AfRGM) Orseolia oryzivora Harris & Gagné (Diptera: Cecidomyiidae) is a major insect pest of lowland rice ecology in Africa. Heavy yield losses have been recorded in many farmers’ rice fields. Use of synthetic insecticides has fostered environmental and human health concern that initiates a search for alternative control measures such as Entomopathogenic fungi (EPF) – Beauveria bassiana and Metarhizium anisopliae. The experiment was laid out on completely randomised design (CRD) with three replications. The study showed M. anisopliae IC30 had the greatest control effect on adult AfRGM with 90.58% of non-infested tillers. The percentage of non-infested tiller advantage over the control followed the same trend with M. anisopliae IC30 having the greatest value of 50.72%. Tiller infestation had significant negative correlation with chlorophyll content, leaf breadth and grain number.

QTL-seq reveals a major root-knot nematode resistance locus on chromosome 11 in rice (Oryza sativa L.)

The root-knot nematode Meloidogyne graminicola is a serious pest in rice affecting production in many rice growing areas. Natural host resistance is an attractive control strategy because the speed of the parasite’s life cycle and the broad host range it attacks make other control measures challenging. Although resistance has been found in the domesticated African rice Oryza glaberrima and the wild rice species O. longistaminata, the introgression of resistance genes to Asian rice O. sativa is challenging. Resistance due to a major gene in O. sativa would greatly aid breeding. Recently two accessions resistant to M. graminicola have been identified in a screen of 332 diverse O. sativa cultivars. In this study, these two resistant cultivars, LD 24 (an indica from Sri Lanka) and Khao Pahk Maw (an aus from Thailand), were crossed with a moderately susceptible cultivar, Vialone Nano (a temperate japonica from Italy). Approximately 175 F2 progeny of both populations were screened for susceptibility to M. graminicola infection. Between 20 and 23 individuals with highest and lowest galls per plants were pooled to make susceptible and resistant bulks which were sequenced to conduct bulked segregant analysis using the QTL-seq method. This revealed a nematode resistance locus from 23 Mbp to the bottom of rice chromosome 11 in both crosses suggesting a rare introgression of the same locus is responsible for resistance in both cultivars. While this information can be used in marker-assisted breeding, analysis of available SNP data revealed candidate loci and genes worthy of further investigation for gene identification.