Patterns of nuclear and chloroplast genetic diversity and structure of manioc along major Brazilian Amazonian rivers

Background and Aims Amazonia is a major world centre of plant domestication, but little is known about how the crops were dispersed across the region. Manioc (Manihot esculenta) was domesticated in the south-western Amazon basin, and is the most important staple food crop that originated in Amazonia. Current contrasting distributions may reflect distinct histories of dispersal of bitter and sweet manioc landraces. To produce new insights into the evolutionary history of the crop, we investigated the contemporary genetic diversity and structure of bitter and sweet manioc along major Amazonian rivers. Methods The patterns of genetic structure and diversity of wild and cultivated sweet and bitter manioc with four chloroplast and 14 nuclear microsatellite markers were evaluated. Results were interpreted in terms of the crop's dispersal. Key results No phylogeographic patterns among rivers were detected, and genetic structure among rivers was confounded by the bitter-sweet divergence. However, differences in the distribution of nuclear diversity and somewhat distinctive patterns of genetic structure across rivers were observed within bitter and sweet manioc. Conclusions Various pre-Columbian and post-European conquest events in the history of Amazonian occupation may explain the absence of clearer patterns of genetic structure. However, the wide distribution of the most common chloroplast haplotype agrees with an early dispersal of manioc across Brazilian Amazonia. Furthermore, differences in genetic structure and in the spatial distribution of genetic diversity suggest that bitter and sweet manioc had distinct dispersal histories. Knowledge about how prehistoric and contemporary Amazonian peoples manage their crops is valuable for the maintenance and conservation of the impressive diversity of their native crops. © 2018 The Author(s). Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved

Spatial genetic structure, genetic diversity and pollen dispersal in a harvested population of Astrocaryum aculeatum in the Brazilian Amazon

Abstract\ud \ud Background\ud \ud Astrocaryum aculeatum is a palm tree species native to the tropical regions of South America, exploited commercially by local farmers for the pulp extracted from its fruits. The objective of this research was to compare the genetic diversity between adult plants and seedlings from open-pollinated seeds, quantify the pollen flow and dispersal, the spatial genetic structure, and the effective size of a population that has been continuously harvested for its fruits. The study was carried out in a natural population of A. aculeatum distributed over approximately 8 ha in the State of Amazonas (Brazil), separated by 400 m from the closest neighboring population. In total, 112 potential pollen donors, 12 mother plants and 120 offspring were mapped and genotyped.\ud \ud \ud Results\ud Genetic diversity was high for parents and the offspring. The fixation indexes for adults (F = -0.035) and offspring (F = -0.060) were negative and not significant. A significant spatial genetic structure was detected for the adult plants (up to the distance of 45 m) indicating short-distance seed dispersal. Paternity analysis detected 9.2 % of pollen immigration and the average distance of pollination within the population was 81 m. The average effective pollination neighborhood area between plants was 1.51 ha.\ud \ud \ud Conclusions\ud Our results indicate that substantial introduction of new alleles has occurred in the population through pollen immigration, contributing to the maintenance of genetic diversity. Conservation efforts aimed at maintaining the gene pool of the current population or establishing new populations should utilize offspring from mother plants selected to be spaced by at least 50 m to prevent collecting seeds from relatives.The authors thank the Fundação de Amparo à Pesquisa do Estado do Amazonas\ud (FAPEAM) for a scholarship to SLFR and supporting the project "Publicação de\ud Artigos Científicos em Genética e Melhoramento de Plantas" - PAPAC (process\ud 062.02685 / 2014); the Conselho Nacional de Desenvolvimento Científico e\ud Tecnológico (CNPq) for scholarships to AMS, MTGL, EAV; the Embrapa Amazônia\ud Ocidental through the project “Pesquisa, desenvolvimento e inovação em\ud palmáceas para a produção de óleo e aproveitamento econômico de coprodutos\ud e resíduos – PROPALMA” (process 01.10.0343.00); and the Coordenação\ud de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) - Programa de\ud Excelência Acadêmica (Proex) of the Genetics Department of ESALQ-USP for\ud supporting this research; and the Laboratório Temático de Biologia Molecular -\ud Instituto Nacional de Pesquisas da Amazônia (LTBM – INPA) for its facilities. The\ud authors also thank Mrs. Angela Mary Wray for the great contribution in the English\ud language and two anonymous reviewers for the excellent suggestions in the\ud manuscript

Development of microsatellite loci to characterize the genetic diversity of annatto (Bixa orellana L.) accessions

O objetivo do presente projeto foi caracterizar a diversidade genética de 63 acessos de urucum (Bixa orellana), mantidos no Banco de Germoplasma do Instituto Agronômico (IAC), a partir da utilização de marcadores microssatélites. Para tanto foram desenvolvidos locos microssatélites para a espécie por meio de uma biblioteca genômica enriquecida. A partir da biblioteca genômica desenvolvida foram obtidas e sequenciadas um total de 84 colônias. Deste total, foram encontrados microssatélites em 57 colônias, o que representa 67,9% de enriquecimento. Foram identificados 70 microssatélites, sendo que destes, foram desenhados e selecionados um total de 31 iniciadores. Dentre estes, 25 iniciadores apresentaram produtos de amplificação, sendo 15 (60%) monomórficos para o grupo de indivíduos estudados. A caracterização dos acessos presentes no germoplasma de urucum, com os 10 iniciadores polimórficos permitiu identificar 38 alelos polimórficos, variando de 2 a 6 alelos por loco, obtendo-se uma média de 3,8 alelos por loco. Os valores de heterozigosidade esperada (He) variaram de 0,464 a 0,765, com média de 0,572. Por outro lado, a heterosigozidade observada (Ho) variou de 0 a 0,744, com média de 0,362. A partir dos índices de fixação de Wright foi possível inferir a taxa aparente de cruzamento, que neste caso foi de 0,39. Os valores de PIC observados neste estudo variaram de 0,359 a 0,725, com média de 0,497. Os marcadores BorB4, BorG3, BorC12, BorG4, BorA2 e BorF5_2 demonstraram ser moderadamente informativos enquanto os iniciadores BorB10, BorB12, BorF1 e BorE7 foram considerados marcadores altamente informativos. A análise de agrupamento para os genótipos avaliados apresentou estruturação em dois grandes grupos, um com acessos da região Norte e outro com os acessos da região Sudeste. Os valores das distâncias de Rogers modificada estimados a partir dos marcadores SSR, variaram de 0 a 0,968, podendo ser identificadas apenas duas duplicatas. O coeficiente de correlação de Pearson (r) entre as distancias genéticas e geográficas obtido com o teste de Mantel foi baixo (r = 0,367), embora significativo (P<0,01). A partir da análise bayesiana realizada pelo software Structure, o conjunto de acessos foi dividido em dois grupos, que coincidiram com os grupos obtidos na análise de agrupamento, demonstrando a consistência dos dados obtidos. Foi possível identificar que o grupo de acessos do Grupo 2 (região Norte) apresenta índices de diversidade superiores ao Grupo 1 (região Sudeste). Os valores médios obtidos de FST (0,183) e GST\' (0,194) indicam a existência de estruturação na amostra total e corrobora os resultados obtidos pela análise de agrupamento, e também com os dados obtidos pela análise de estruturação com o software Structure. Os valores médios de FIT (0,483) e HT\' (0.632) demonstraram a elevada diversidade genética no conjunto de acessos estudado. O fato de que os valores de FIS e GIS sejam maiores do que os valores de FST e GST para praticamente todos os locos, indicam a existência de maior diversidade dentro de cada grupo de acessos do que entre os grupos. A manutenção do banco ativo de germoplasma de urucum possibilita a preservação do patrimônio genético e pode fornecer matéria prima para as atividades de melhoramento e fomento da cadeia produtiva da cultura.The aim of this project was to characterize the genetic diversity of 63 accessions of annatto (Bixa orellana), maintained at the Germplasm Bank of the Agronomic Institute (IAC), using microsatellite markers. For that purpose, microsatellite loci have been developed for this species through an enriched DNA genomic library. From the genomic library developed a total of 84 colonies were obtained and sequenced. Microsatellites were found in 57 colonies, representing 67.9% of enrichment. Seventy microsatellites were identified and, from those, a total of 31 primers were selected. Among these, 25 primers showed amplification products and 15 (60%) were monomorphic for the group of accessions studied. The germplasm accessions characterization with the 10 polymorphic primers showed 38 polymorphic alleles, ranging from 2 to 6 alleles per locus, yielding an average of 3.8 alleles per locus. Values of expected heterozygosity (He) ranged from 0.464 to 0.765, averaging 0.572. Moreover, the observed heterozygosity (Ho) ranged from 0 to 0.744, averaging 0.362. From the Wright\'s fixation index, it was possible to infer the apparent outcrossing rate, which in this case was 0.39. The PIC values observed in this study ranged from 0.359 to 0.725, averaging 0.497. The markers BorB4, BorG3, BorC12, BorG4, and BorA2 BorF5_2 were shown to be moderately informative, and the primers BorB10, BorB12, BorF1 BorE7 were considered highly informative markers. The cluster analysis for the genotypes showed structuring into two major groups, one with the accessions from the North and another with the accessions from the Southeast. The values of the modified Rogers distances estimated from the SSR markers ranged from 0 to 0.968, and only two duplicates were identified. The Pearson correlation coefficient (r) between genetic and geographic distances obtained with the Mantel test was low (r = 0.367), although significant (P <0.01). From the Bayesian analysis performed by the software Structure, the accessions were divided into two groups, which coincided with the groups obtained from the cluster analysis, demonstrating the consistency of the results obtained. It was possible to identify that the accessions in Group 2 (Northern region) present diversity indexes higher than Group 1 (Southeast). The average values of FST (0.183) and GST\' (0.194) indicate the existence of genetic structure in the total sample and confirms the results obtained in the cluster analysis, and also with the data obtained in the structure analysis using the software Structure. The average values of FIT (0.483) and HT\' (0.632) demonstrated the high genetic diversity among the accessions studied. The fact that the FIS and GIS values are higher than the values of FST and GST for almost all loci indicates that diversity is higher within each group than between groups of accessions. Maintaining the annatto germplasm bank enables the preservation of genetic resources and can provide material for breeding activities and improvements in the crop production chain

Estrutura genética, sistema reprodutivo e domesticação de urucum (Bixa orellana L.) utilizando marcadores moleculares

Plant domestication is an evolutionary process that leads to several modifications in plants to increase adaptation to cultivation and utilization by humans. These modifications may decrease the fitness of plants in the wild habitat but increase it for human exploitation. Annatto (Bixa orellana L.) is a shrubby plant domesticated in Amazonia from wild annatto (Bixa orellana var. urucurana) populations. This thesis presents a more in-depth understanding of the domestication, mating system and genetic diversity and structure of annatto and its wild ancestor in Brazil. In the first study, a new set of 32 microsatellite loci isolated from a microsatellite-enriched genomic library was developed, of which 12 were polymorphic in populations of both cultivated and wild annatto. In the second study, the genetic diversity and structure of wild annatto populations in Brazilian Amazonia were characterized with 16 microsatellite markers. High population structure and positive correlation between genetic and geographic distances were found, suggesting that genetic differentiation might be caused by geographic isolation. Additionally, Ecological Niche Modeling was used to characterize the potential geographical range of this variety in northern South America and detected that South Rondônia, Madre di Dios River basin, Llanos de Mojos, Llanos de Orinoco and eastern Ecuador are highly suitable areas for wild annatto to occur, providing additional targets for future exploration and conservation. In the third study, 16 microsatellite loci and four phytochemical compounds were used to evaluate the genetic diversity of 63 accessions from the annatto germplasm bank at the Agronomic Institute (IAC). In both molecular and phytochemical analysis the results tended to separate the accessions from Rondônia, northern Brazil, from the Southwestern accessions. Rondônia accessions showed higher values for all the phytochemical compounds and higher levels of genetic diversity. Some accessions presented bixin levels well above the average and are promising materials to be used in genetic improvement programs. In the fourth study, 12 microsatellite loci were used to determine the mating system of a cultivated population of annatto from Rondon do Pará, PA. Multilocus outcrossing rate indicated a mixed mating system for this population. Biparental inbreeding also contributed to the selfing rate in this population. Crossings among related individuals were also observed. Due to this mixed breeding system, the collection of open-pollinated seeds for plant breeding or conservation purposes should include at least 60 plants to ensure a representative sample. In the fifth study, the amount and distribution of genetic diversity among samples of cultivated annatto from homegardens of riverside communities along the major rivers in Brazilian Amazonia, and from farmer´s fields along highways, in the States of Rondônia and Pará, and Southeastern Brazil was characterized. The samples collected presented moderate levels of genetic diversity, and moderate to high levels of admixture between geographic groups, occurring mainly due to exchange of seeds among farmers. However, cluster and Bayesian analyses showed a tendency to group samples based on their geographic origin. Isolation by distance was observed, according to Mantel\'s test. In the last study, wild and cultivated annatto samples from Brazilian Amazonia were compared using 16 microsatellite loci and two cpDNA regions. A clear separation between wild and cultivated annatto, supported by high values of FST in both analyses was observed. Wild samples presented higher rates of diversity in relation to cultivated, partly because these populations did not suffer anthropic selection, as in the cultivated varieties. The data suggest the existence of genetic relationship between wild and cultivated annatto, indicated by moderate levels of gene flow. The results also showed the proximity between groups of cultivated and wild accessions from Rondônia and the Madeira River basin. This proximity provides indications that annatto started its domestication in this area from B. orellana var. urucurana.Domesticação de plantas é um processo evolutivo que pode gerar uma série de modificações nas plantas para aumentar a adaptação para o cultivo e utilização pelos humanos. Estas modificações podem diminuir a aptidão das plantas no habitat selvagem, porém, aumentando sua aptidão para exploração humana. Urucum (Bixa orellana L.) é uma planta arbustiva domesticada na Amazônia a partir de populações de Bixa orellana var. urucurana. Esta tese apresenta um entendimento mais aprofundado sobre a domesticação, sistema reprodutivo e diversidade genética e estrutura de urucum e seu ancestral selvagem no Brasil. No primeiro estudo, um novo conjunto de 32 locos microssatélites foram isolados a partir de uma biblioteca genômica enriquecida com microssatélites, dos quais 12 foram polimórficos em populações de urucum selvagem e cultivado. No segundo estudo, a diversidade e estrutura genética de populações selvagens de urucum na Amazônia brasileira foram caracterizadas usando 16 marcadores microssatélites. Elevada estrutura populacional, e correlações positivas entre distancias genéticas e geográficas foram observadas, sugerindo que a diferenciação genética é resultante de isolamento geográfico. Adicionalmente, Modelagem de Nicho Ecológico foi utilizada para caracterizar a distribuição potencial desta variedade no norte da América do Sul e observamos que o Sul de Rondônia, a bacia do rio Madre de Dios, os Llanos de Mojos e de Orinoco e oeste do Equador são áreas de alta probabilidade de ocorrência de urucum selvagem, fornecendo informações importantes para novas amostragens e conservação. No terceiro estudo, 16 locos de microssatélites e quatro compostos fitoquímicos foram utilizados para avaliar a diversidade genética de 63 acessos do banco de germoplasma de urucum do Instituto Agronômico (IAC). Em ambas as análises, houve uma tendência de separação dos acessos de Rondônia, norte do Brasil, dos acessos do Sudeste. Os acessos de Rondônia apresentaram elevados valores para todos os compostos fitoquímicos e também apresentaram altos níveis de diversidade genética. Alguns acessos apresentaram níveis de bixina acima da média e são considerados materiais promissores para uso em programas de melhoramento genético de urucum. No quarto estudo, 12 locos microssatélites foram utilizados para determinar o sistema de cruzamento de uma população de urucum de Rondon do Pará, PA. A taxa de cruzamento multilocos indicou um sistema misto de cruzamento para esta população. A endogamia biparental também contribuiu para a taxa de autofecundação. Cruzamentos entre indivíduos aparentados também foram observados. Devido ao sistema misto, a coleta de sementes de polinização aberta para fins de conservação e melhoramento genético deve incluir pelo menos 60 plantas para assegurar uma amostragem representativa. No quinto estudo, a distribuição da diversidade genética entre amostras de urucum cultivado de quintais de comunidades ribeirinhas dos principais rios da Amazônia Brasileira, além de plantações ao longo das rodovias dos estados do Rondônia e Pará, além do Sudeste do Brasil foi caracterizada. As amostras coletadas apresentaram moderados níveis de diversidade genética e moderados a altos níveis de fluxo gênico entre os grupos geográficos, principalmente devido ao intercambio de semente entre agricultores. Contudo, análises Bayesianas e de agrupamento indicaram uma tendência de agrupamento baseado na origem geográfica das amostras. Isolamento por distância também foi observado de acordo com o teste de Mantel. No último estudo, amostras de urucum selvagem e cultivado da Amazônia brasileira foram comparados utilizando 16 locos microssatélites e duas regiões de DNA cloroplastidial. Uma clara separação entre cultivados e selvagens, suportada por altos valores de FST em ambas as análises foi observado. Amostras selvagens apresentaram altas taxas de diversidade em relação aos cultivados, parcialmente por não sofrem seleção antrópica como acontece nas variedades cultivadas. Os dados sugerem a existência de relações genéticas entre urucum selvagem e cultivado, indicado por moderados níveis de fluxo gênico. Os resultados também demonstraram a proximidade entre grupos de urucum selvagem e cultivados de Rondônia e da bacia do Rio Madeira. Esta proximidade fornece indícios que a domesticação de urucum iniciou nesta região a partir de B. orellana var. urucurana

Be-Breeder – an application for analysis of genomic data in plant breeding

Be-Breeder is an application directed toward genetic breeding of plants, developed through the Shiny package of the R software, which allows different phenotype and molecular (marker) analysis to be undertaken. The section for analysis of molecular data of the Be-Breeder application makes it possible to achieve quality control of genotyping data, to obtain genomic kinship matrices, and to analyze genomic selection, genome association, and genetic diversity in a simple manner on line. This application is available for use in a network through the site of the Allogamous Plant Breeding Laboratory of ESALQ-USP (http://www.genetica.esalq.usp.br/alogamas/R.html)

The domestication of annatto (Bixa orellana) from bixa urucurana in Amazonia

Annatto (Bixa orellana) is an important colorant domesticated in theNeotropics, although it is not clear where or from which wild populations. We reviewed the available biological, archaeological, and ethnographic information about annatto, and integrated this with our recent ethnobotanical observations of cultivated and non-cultivated populations in order to evaluate the hypothesis thatwhat is classified as Bixa urucurana is the wild ancestor of cultivated annatto, Bixa orellana. Most B. urucurana populations we found in Amazonia occurred in open forests or anthropogenic landscapes, although never cultivated, and always associated with riparian environments. While cultivated annatto always produces abundant pigment, B. urucurana populations that we observed contained variable amounts of pigment, fromvery little to nearly the amount of cultivated annatto, suggesting gene flow fromcultivated to non-cultivated. Bixa urucurana has indehiscent fruits, which indicate changes in dehiscence during annatto domestication, a notable feature rarely found in other tree species. Local residents identified the non-cultivated populations aswild annatto (urucum bravo), and they emphasized their smaller fruits with less pigment, their spontaneous regeneration, their non-use, and that they hybridize with cultivated annatto. Ethnography identified the symbolic importance of annatto, but an explicit mention of origin only comes from southern Amazonia. Although the oldest annatto archaeological record came from the Caribbean, domestication occurred in northern South America, since B. urucurana does not occur in the Caribbean. Traditional ecological knowledge and morphology identified the close relationship between B. urucurana (never cultivated) and B. orellana (always cultivated). Evidence reported here strongly supports Kuntze's (1925) suggestion that Bixa urucurana Willd. is a variety of B. orellana L., thus identifying the wild ancestor of cultivated annatto. © 2015, by The New York Botanical Garden Press, Bronx, NY 10458-5126 U.S.A

Development and characterization of microsatellite loci for the Neotropical orchid Trichocentrum pumilum

Studies of genetic diversity and structure are key elements in designing effective in situ and ex situ management plans, especially for species experiencing forest fragmentation. To investigate the level of genetic diversity in populations of Trichocentrum pumilum, eight polymorphic microsatellite loci were developed and used for genotyping 96 specimens from four disturbed populations. Low genetic diversity within populations was found (average number of alleles per locus ranging from 3.75 to 4.25, observed and expected heterozygosities from 0.238 to 0.333 and from 0.450 to 0.482, respectively). The fixation index (FIS) ranged from 0.35 to 0.47, with significant values for all populations. No genotypic disequilibrium was detected. A mixed breeding system was found through an apparent outcrossing rate estimate. Our results suggest that these microsatellite loci are suitable for genetic studies of this species, showing low within population genetic diversity and moderate structure for T. pumilum populations