Molecular and morphological diversity of on-farm hazelnut (Corylus avellana L.) landraces from southern Europe and their role in the origin and diffusion of cultivated germplasm

Hazelnut (Corylus avellana L.) is a traditional nut crop in southern Europe. Germplasm exploration conducted on-farm in five countries (Portugal, Spain, Italy, Slovenia, and Greece) identified 77 landraces. The present work describes phenotypic variation in nut and husk traits and investigates genetic relationships using ten simple sequence repeat (SSR) markers among these landraces, 57 well-known references cultivars, and 19 wild accessions. Among the 77 landraces, 42 had unique fingerprints while 35 showed a SSR profile identical to a known cultivar. Among the 42 unique landraces, morphological observations revealed high phenotypic diversity, and some had characteristics appreciated by the market such as nut round and caliber. Analysis of genetic relationships and population structure allowed investigation of the origin and spread of the cultivated germplasm in southern Europe. Our results indicate the existence of three primary centers of diversity in the Mediterranean basin: northwestern Spain (Tarragona) and southern Italy (Campania) in the West and Black Sea (Turkey) in the East. Moreover, the data suggest the existence of secondary gene pools in the Iberian (Asturias) and Italian (Liguria and Latium) Peninsulas, where local varieties were recently domesticated from wild forms and/or from introduced ancient domesticated varieties.     

水稻Rim2/Hipa 超级家族的基因组变异和基于Rim2/Hipa 展示的水稻资源的系统进化和指纹分析

水稻Rim2/Hipa是最近鉴定的一个受逆境诱导的转座因子超级家族.研究表明,Rim2的核心序列在不同来源的水稻材料中存在显著的差异,暗示Rim2家族的长期进化历程.基于Rim2因子间的差异性以及该因子的静止状态,开发出一种利用Rim2因子展示的新的分子指纹技术,可以灵敏地区分不同水稻资源以及它们的遗传关系.仅用5对引物就可以清楚地将53个栽培稻和普通野生稻材料鉴定出来,并可将它们分为不同的系统进化组.研究表明不仅在水稻资源而且在野生稻种质间均存在明显的多样性.野生稻可以被单独分组,或者分散在粳稻中间.这种新的指纹技术还可以将水稻的杂交子代和它们的亲本区分出来,并可用于种子纯度的鉴定,在水稻基因组进化研究、水稻育种和种子生产中有很好的应用前景.     

Unlocking Allelic Diversity for Sustainable Development of Salinity Stress Tolerance in Rice

Rice is a major cereal crop, negatively impacted by soil-salinity, both in terms of plant growth as well as productivity. Salinity tolerant rice varieties have been developed using conventional breeding approaches, however, there has been limited success which is primarily due to the complexity of the trait, low yield, variable salt stress response and availability of genetic resources. Furthermore, the narrow genetic base is a hindrance for further improvement of the rice varieties. Therefore, there is a greater need to screen available donor germplasm in rice for salinity tolerance related genes and traits. In this regard, genomics based techniques are useful for exploring new gene resources and QTLs. In rice, the vast allelic diversity existing in the wild and cultivated germplasm needs to be explored for improving salt tolerance. In the present review, we provide an overview of the allelic diversity in the Quantitative Trait Loci (QTLs) like Saltol, qGR6.2, qSE3 and RNC4 as well as genes like OsHKT1;1, SKC1 (OsHKT1;5/HKT8) and OsSTL1 (salt tolerance level 1 gene) related to salt tolerance in rice. We have also discussed approaches for developing salt-tolerant cultivars by utilizing the effective QTLs or genes/alleles in rice.     

Farmers Drive Genetic Diversity of Thai Purple Rice (Oryza sativa L.) Landraces

Purple or black rice (Oryza sativa L.) is a culturally important germplasm in Asia with a long history of cultivation in northern Thailand. Purple rice is identified by the color of the rice pericarp, which varies from purple to black with the accumulation of phenolic acids, flavonoids, and anthocyanins. In the present study, we assessed molecular variation within and between wetland purple rice landraces germplasm from northern and northeastern Thailand using 12 microsatellite loci. All purple rice varieties surveyed showed high levels of homozygosity within varieties and strong genetic differentiation among varieties, indicating the fixation of genetic differences among them. This pattern is consistent with purple rice farming practices in northern Thailand, where a small portion of harvested seed is selected and replanted based on farmers’ preferences. The reduced genetic diversity and high homozygosity observed for purple rice is also consistent with patterns expected for this inbreeding crop. Genetic differentiation among the varieties showed some degree of structuring based on their geographical origin. Taken together, these data highlight that the genetic diversity and structure of wetland purple rice landraces is shaped by farmer utilization and cultivation through local cultural practices, and that conservation should focus on ex situ conservation across its cultivation range, along with on-farm, in situ conservation based on farmers’ seed-saving practices. In situ conservation may prove especially valuable for preserving the genetic identity of local varieties and promote adaptation to local environments.

     

Genetic analysis of Indian aromatic and quality rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) germplasm using panels of fluorescently-labeled microsatellite markers

Genetic relationships among Indian aromatic and quality rice (Oryza sativa) germplasm were assessed using 30 fluorescently labeled rice microsatellite markers. The 69 rice genotypes used in this study included 52 Basmati and other scented/quality rice varieties from different parts of India and 17 indica and japonica varieties that served as controls. A total of 235 alleles were detected at the 30 simple sequence repeat (SSR) loci, 62 (26.4%) of which were present only in Basmati and other scented/quality rice germplasm accessions. The number of alleles per locus ranged from 3 to 22, with an average of 7.8, polymorphism information content (PIC) values ranged from 0.2 to 0.9, with an average of 0.6, and the size range between the smallest and the largest allele for a given microsatellite locus varied between 3 bp and 68 bp. Of the 30 SSR markers, 20 could distinguish traditional Basmati rice varieties, and a single panel of eight markers could be used to differentiate the premium traditional Basmati, cross-bred Basmati, and non-Basmati rice varieties having different commercial value in the marketplace. When estimates of inferred ancestry or similarity coefficients were used to cluster varieties, the high-quality Indian aromatic and quality rice genotypes could be distinguished from both indica and japonica cultivars, and crossbred varieties could be distinguished from traditional Basmati rices. The results indicate that Indian aromatic and quality germplasm is genetically distinct from other groups within O. sativa and is the product of a long, independent pattern of evolution. The data also suggest that there is scope for exploiting the genetic diversity of aromatic/quality rice germplasm available in India for national Basmati rice breeding programs.Electronic Supplementary Material Supplementary material is available for this article at .     

水稻SSR标记的遗传多样性研究进展

本文从SSR标记优点和适用于研究水稻遗传多样性入手,综述了SSR标记在水稻核心种质构建与评价、遗传结构、稻种起源演化等方面的研究进展。总结了水稻遗传多样性的地带性特征（云南是中国稻种资源的最大遗传多样性中心和优异种质的富集地;西南稻区粳稻品种遗传多样性最丰富;南方稻区粳稻品种的遗传多样性高于北方粳稻遗传多样性）、遗传多样性与生态地理位置密切相关、目前水稻品种遗传基础狭窄、多样性降低等特征,分析了遗传多样性成因及影响因素,特别指出了育种行为对遗传多样性的影响,并针对当前水稻品种遗传多样性较低的问题提出了对策。     

Assessment of genetic diversity in Italian rice germplasm related to agronomic traits and blast resistance (<Emphasis Type="Italic">Magnaporthe</Emphasis><Emphasis Type="Italic">oryzae</Emphasis>)

Italy is the only country in Europe with a significant land area used for rice production. In this paper, the genetic diversity of 172 national varieties and 47 foreign accessions (ITALORYZA collection) was investigated using a set of neutral markers evenly distributed throughout the 12 chromosomes. Out of the 218 alleles detected in our analysis, 17 and 29% were specific to the Italian and foreign accessions, respectively. From the neighbour-joining tree generated, six sub-groups of temperate japonica germplasm were identified. Plant height and grain type measured in the source collection were fitted to the phylogenetic tree, along with the period of variety registration. This integrated genotype–phenotype analysis revealed that specific sub-groups are characterized by uniform classes of grain type, or by similar plant size, or by period of release in the market. The whole collection was also evaluated for leaf blast resistance by inoculating the plants with three strains of Magnaporthe oryzae, representing the pathogen genetic diversity existing in Italy. Only 15 out of 172 Italian accessions (8.7%) were resistant to all three fungal strains. The correlation between genotype and leaf blast phenotype revealed that the most highly resistant Italian varieties are included in a single germplasm sub-group derived from US varieties. This study represents the starting point for carrying out detailed phenotype–genotype whole-genome association studies and identification of the genetic basis of important agronomic traits for rice cultivation in temperate climates.     

Genetic differentiation among Sri Lankan traditional rice (Oryza sativa) varieties and wild rice species by AFLP markers

Traditional rice varieties are one important component of the biodiversity of Sri Lanka. However, no proper studies have been performed on genetic diversity of the Sri Lankan traditional rice varieties used in our breeding programs. In the present study, the genetic diversity of 46 traditional rice (Oryza sativa) varieties and 5 wild rice species is assesed using Amplified Fragment Length Polymorphism (AFLP) markers. Ten primer combinations generated a total of 784 fragments. Of these, 772 fragments were polymorphic (98.4%). UPGMA analysis based on Jaccard's similarity separated the accessions into four major clusters. A cophenetic correlation with r=0.786 strongly supported the clustering pattern of UPGMA dendrogram. A principal coordinate analysis (PCoA) also confirmed the UPGMA clusters. Accessions referred to the same cluster showed similar morphological characteristics (e.g. height, grain colour etc.) while accessions which are known to be morphologically distinct appeared genetically separated. In addition, the clustering pattern distinctly separated lowland and upland rice varieties. This genetic diversity assessment at the molecular level provides reliable information for selection of germplasm in the development of new rice varieties and in conservation of traditional rice genetic resources.     

Rice genetic resources: history,conservation, investigative characterization and use in Japan

Rice has been grown in Japan for about 3000 years. Although both japonica and indica varieties have been grown in Japan, now japonica rices are grown. Japanese rice breeding has used an ecological breeding approach. While emphasis in rice breeding in the 1940's and 1950's focussed on yield in recent decades quality has been of major importance. Consumer preference and name recognition of high quality varieties, such as Koshihikari, has resulted in slow acceptance of new varieties.Rice germplasm was systematically collected throughout Japan between 1962 and 1963. Subsequent acquisition and collecting, in Japan and other countries, has resulted in 28,000 accessions being conserved in the National Genebank, based at the National institute of Agrobiological Resources (NIAR).Research on genetic diversity of rice using a range of techniques, for example esterase isozymes, has revealed clinal variation in rice radiating from the center of diversity of rice in and around southwest China. Newly found genes in traditional rice germplasm, such as genes for non-elongating mesocotyl, are now routinely identified on the rice genome. Pioneering studies on eco-genetic differentiation of species in the genus Oryza in Japan has revealed much about the complex genepool for which rice evolved.Pest and disease resistance sources, particularly to blast, bacterial blight and brown plant hopper, from many countries have been incorporated into Japanese varieties. Cold tolerance at the booting stage was found in the Indonesian variety Silewah. In the future in characterisation of rice germplasm and interaction between rice germplasm specialists and rice molecular scientists, both in Japan and internationally, will be corner stones to securing rice genetic diversity and rice improvement in the next century.     

Conservation of rice genetic resources: the role of the International Rice Genebank at IRRI

Rice genetic resources, comprising landrace varieties, modern and obsolete varieties, genetic stocks, breeding lines, and the wild rices, are the basis of world food security. The International Rice Genebank at the International Rice Research Institute in the Philippines conserves the largest and most diverse collection of rice germplasm. The facilities of the genebank ensure the long-term preservation of this important diversity. In field research, factors that affect long-term viability of rice seeds have been identified, leading to the introduction of modified practices for germplasm multiplication and regeneration. The value of conserved germplasm can be assessed in terms of useful traits for rice breeding and the economic impact that germplasm utilization has on rice production and productivity. The application of molecular markers is changing perspectives on germplasm management. International policies affecting access to and use of rice germplasm are discussed.     

Genetic Diversity and Population Structure in Aromatic and Quality Rice (Oryza sativa L.) Landraces from North-Eastern India

The North-eastern (NE) India, comprising of Arunachal Pradesh, Assam, Manipur, Meghalaya, Mizoram, Nagaland, Sikkim and Tripura, possess diverse array of locally adapted non-Basmati aromatic germplasm. The germplasm collections from this region could serve as valuable resources in breeding for abiotic stress tolerance, grain yield and cooking/eating quality. To utilize such collections, however, breeders need information about the extent and distribution of genetic diversity present within collections. In this study, we report the result of population genetic analysis of 107 aromatic and quality rice accessions collected from different parts of NE India, as well as classified these accessions in the context of a set of structured global rice cultivars. A total of 322 alleles were amplified by 40 simple sequence repeat (SSR) markers with an average of 8.03 alleles per locus. Average gene diversity was 0.67. Population structure analysis revealed that NE Indian aromatic rice can be subdivided into three genetically distinct population clusters: P1, joha rice accessions from Assam, tai rices from Mizoram and those from Sikkim; P2, chakhao rice germplasm from Manipur; and P3, aromatic rice accessions from Nagaland. Pair-wise F_ST between three groups varied from 0.223 (P1 vs P2) to 0.453 (P2 vs P3). With reference to the global classification of rice cultivars, two major groups (Indica and Japonica) were identified in NE Indian germplasm. The aromatic accessions from Assam, Manipur and Sikkim were assigned to the Indica group, while the accessions from Nagaland exhibited close association with Japonica. The tai accessions of Mizoram along with few chakhao accessions collected from the hill districts of Manipur were identified as admixed. The results highlight the importance of regional genetic studies for understanding diversification of aromatic rice in India. The data also suggest that there is scope for exploiting the genetic diversity of aromatic and quality rice germplasm of NE India for rice improvement.     

Single-seeded InDel fingerprints in rice:An effective tool for indica-japonica rice classification and evolutionary studies

Asian cultivated rice(Oryza sativa L.),an important cereal crop worldwide,was domesticated from its wild ancestor 8000 years ago.During its long-term cultivation and evolution under diverse agroecological conditions, Asian cultivated rice has differentiated into indica and japonica subspecies.An effective method is required to identify rice germplasm for its indica and japonica features,which is essential in rice genetic improvements.We developed a protocol that combined DNA extraction from a single rice seed and the insertion/deletion(InDel) molecular fingerprint to determine the indica and japonica features of rice germplasm.We analyzed a set of rice germplasm,including 166 Asian rice varieties,two African rice varieties,30 accessions of wild rice species,and 42 weedy rice accessions,using the single-seeded InDel fingerprints(SSIF).The results show that the SSIF method can efficiently determine the indica and japonica features of the rice germplasm.Further analyses revealed significant indica and japonica differentiation in most Asian rice varieties and weedy rice accessions.In contrast,African rice varieties and nearly all the wild rice accessions did not exhibit such differentiation.The pattern of cultivated and wild rice samples illustrated by the SSIF supports our previous hypothesis that indica and japonica differentiation occurred after rice domestication under different agroecological conditions.In addition,the divergent pattern of rice cultivars and weedy rice accessions suggests the possibility of an endoferal origin(from crop)of the weedy rice included in the present study.     

Assessment of Genetic Diversity Among Rice Genotypes with Differential Adaptations to Salinity Using Physio-morphological and Molecular Markers

Breeding for salt tolerance using traditional screening and selection methods have been limited by the complex and polygenic nature of salt tolerance trait. This study was designed to evaluate some of the premium Basmati rice varieties for salt tolerance and to characterize genetic diversity among the rice varieties with different adaptations to saline soils using microsatellite (SSR) and ISSR markers. Plants of nine rice varieties including salt tolerant, salt sensitive and traditional Basmati, were grown in hydroponics using Yoshida solution containing 0 (control, pH 5.0) and 30 mM NaCl (Electrical conductivity 4.8 d/S, pH 5.0) and assessed for salinity tolerance on 1–9 scale as per IRRI standard evaluation system using seedling growth parameters, visual salt injuries and Na-K ratio. Physio-morphological studies showed that traditional Basmati rice varieties (Basmati 370 and HBC19) were more sensitive than the salt sensitive control variety, MI-48. SSR as well as ISSR marker systems generated higher levels of polymorphism and could distinguish between all the 9 rice cultivars. A total of 299 (225 polymorphic) and 437 (430 polymorphic) bands were detected using 28 UBC ISSR primers and 100 welldistributed mapped SSR markers, respectively. ISSR and SSR marker data-sets showed moderate levels of positive correlation (Mantel test, r = 0.43). The ISSR and SSR marker data analyzed using clustering algorithms showed two distinct clusters separating the Basmati (Basmati 370, HBC19 and CSR-30) from other non-aromatic indica (IR36, Pokkali, CSR10 and MI-48) rice varieties indicating greater divergence between Basmati and non-aromatic indica rice genotypes. Marker analysis showed a close relationship among the two traditional (Basmati 370 and HBC19) and cross-bred (CSR30) Basmati rice varieties and greater diversity between the two salt-tolerant genotypes, Pokkali and BR4-10.     

复杂的农业景观系统中植物物种多样性的评价方法-PLEC农业生物多样性指导小组（PLEC-BAG）对资料搜集与分析的指南

农业生物多样性已成为国际关注的热点领域之一。本文根据郭辉军等中国专家提出的农业生物多样性评价（Agobiodiyersity Assessment,ABA）方法为基础,提出了景观水平农业生物多样性评价相关术语、基本用词的定义,一系列用于搜集PLEC植物物种多样性核心资料的基本原理与实用指南,以及资料分析与报告指南,以指导全球环境基金/联合国环境署/联合国大学PLEC项目计划20多个国家的研究。图表可于实地调查时用于搜集及分析核心资料;这些资料最终将会包括于PLEC物种多样性数据库。正文则提供辅助的咨询解释。     

The International Rice Information System. A platform for meta-analysis of rice crop data

Ambiguous germplasm identification; difficulty in tracing pedigree information; and lack of integration between genetic resources, characterization, breeding, evaluation, and utilization data are constraints in developing knowledge-intensive crop improvement programs. To address these constraints, the International Crop Information System (www.icis.cgiar.org), a database system for the management and integration of global information on genetic resources and crop improvement for any crop, was developed by genetic resource specialists, crop scientists, and information technicians associated with the Consultative Group for International Agricultural Research and collaborative partners. The International Rice Information System (www.iris.irri.org) is the rice (Oryza species) implementation of the International Crop Information System. New components are now being added to the International Rice Information System to handle the diversity of rice functional genomics data including genomic sequence data, molecular genetic data, expression data, and proteomic information. Users access information in the database through stand-alone programs and Web interfaces, which offer specialized applications and customized views to researchers with different interests.     

Genomics-based precision breeding approaches to improve drought tolerance in rice

Rice (Oryza sativa L.), the major staple food crop of the world, faces a severe threat from widespread drought. The development of drought-tolerant rice varieties is considered a feasible option to counteract drought stress. The screening of rice germplasm under drought and its characterization at the morphological, genetic, and molecular levels revealed the existence of genetic variation for drought tolerance within the rice gene pool. The improvements made in managed drought screening and selection for grain yield under drought have significantly contributed to progress in drought breeding programs. The availability of rice genome sequence information, genome-wide molecular markers, and low-cost genotyping platforms now makes it possible to routinely apply marker-assisted breeding approaches to improve grain yield under drought. Grain yield QTLs with a large and consistent effect under drought have been indentified and successfully pyramided in popular rice mega-varieties. Various rice functional genomics resources, databases, tools, and recent advances in “-omics” are facilitating the characterization of genes and pathways involved in drought tolerance, providing the basis for candidate gene identification and allele mining. The transgenic approach is successful in generating drought tolerance in rice under controlled conditions, but field-level testing is necessary. Genomics-assisted drought breeding approaches hold great promise, but a well-planned integration with standardized phenotyping is highly essential to exploit their full potential.     

Genetic variation of a global germplasm collection of chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.) including Italian accessions at risk of genetic erosion

Chickpea (Cicer arietinum L.) is one of the most important legumes worldwide. We addressed this study to the genetic characterization of a germplasm collection from main chickpea growing countries. Several Italian traditional landraces at risk of genetic erosion were included in the analysis. Twenty-two simple sequence repeat (SSR) markers, widely used to explore genetic variation in plants, were selected and yielded 218 different alleles. Structure analysis and hierarchical clustering indicated that a model with three distinct subpopulations best fits the data. The composition of two subpopulations, named K1 and K2, broadly reflects the commercial classification of chickpea in the two types desi and kabuli, respectively. The third subpopulation (K3) is composed by both desi and kabuli genotypes. Italian accessions group both in K2 and K3. Interestingly, this study highlights genetic distance between desi genotypes cultivated in Asia and Ethiopia, which respectively represent the chickpea primary and the secondary centres of diversity. Moreover, European desi are closer to the Ethiopian gene pool. Overall, this study will be of importance for chickpea conservation genetics and breeding, which is limited by the poor characterization of germplasm collection.     

Abundant Within-varietal Genetic Diversity in Rice Germplasm from Yunnan Province of China Revealed by SSR Fingerprints

In order to estimate genetic diversity of rice (Oryza sativa L.) germplasm in Yunnan Province of China, 60 varieties from different regions were analyzed by microsatellite (SSR) fingerprints. Nine selected SSR primer pairs amplified a total of 55 alleles from these varieties, and high genetic diversity (0.706) was found, although it was not evenly distributed across the regions. Marked genetic variation was detected within the traditional varieties. A UPGMA dendrogram based on SSR polymorphism indicated a great variation among the rice varieties, with coefficients ranging between 0.229 and 1.000. The formation of the rice diversity pattern in Yunnan is associated with natural conditions and especially with diverse cultural demands and farming styles. Strategic conservation of rice germplasm in Yunnan is important, and this could be implemented by collecting varieties across geographic regions with sufficient individuals within the same varieties. Effective rice conservation should also consider cultural aspects during collection.     

东北三省2021年水稻审定品种的核心亲本遗传多样性与籼粳分化北大核心CSCD

为了阐明东北三省近年育成水稻品种的遗传基础,该研究通过对2021年育成的256个水稻新品种的系谱进行分析,确定了20个材料为育种核心亲本,并利用44对多态性SSR分子标记和34对InDel籼粳特异性分子标记分析了它们的遗传多样性和籼粳分化程度。结果表明:(1)SSR标记能够高效评价参试材料的多样性水平,共检测到130个等位基因,每个位点检测等位基因(Na)变异为2~5个,平均为2.95个;基因多样性指数(H)变异为0.10~0.75,平均为0.41;多态性信息含量(PIC)变异为0.09~0.70,平均为0.36。(2)遗传多样性分析表明,东北三省20份水稻核心亲本的整体多样性水平较低,黑龙江省核心亲本多样性略高于辽宁省和吉林省。(3)NJ聚类分析表明,20份核心亲本可以划分为3个类群,分别为辽宁类群、吉林类群和黑龙江类群。(4)遗传结构分析表明,20份核心亲本可划分为2个稳定的类群,其中辽宁省核心亲本均处于A类群,黑龙江省核心亲本均处于B类群。(5)籼粳分化分析表明,20份核心亲本遗传背景均为粳稻类型,但都含有少数籼型位点,其籼型基因频率F在0.01~0.19范围内。研究认为,东北三省水稻育种核心亲本遗传基础狭窄、使用频率较高是导致育成品种遗传多样性水平较低的可能原因,在育种中亟待挖掘新种质、引入新资源,丰富遗传背景。     

The Birth of a Black Rice Gene and Its Local Spread by Introgression

The origin and spread of novel agronomic traits during crop domestication are complex events in plant evolution. Wild rice (Oryza rufipogon) has red grains due to the accumulation of proanthocyanidins, whereas most cultivated rice (Oryza sativa) varieties have white grains induced by a defective allele in the Rc basic helix-loop-helix (bHLH) gene. Although the events surrounding the origin and spread of black rice traits remain unknown, varieties with black grains due to anthocyanin accumulation are distributed in various locations throughout Asia. Here, we show that the black grain trait originated from ectopic expression of the Kala4 bHLH gene due to rearrangement in the promoter region. Both the Rc and Kala4 genes activate upstream flavonol biosynthesis genes, such as chalcone synthase and dihydroflavonol-4-reductase, and downstream genes, such as leucoanthocyanidin reductase and leucoanthocyanidin dioxygenase, to produce the respective specific pigments. Genome analysis of 21 black rice varieties as well as red- and white-grained landraces demonstrated that black rice arose in tropical japonica and its subsequent spread to the indica subspecies can be attributed to the causal alleles of Kala4. The relatively small size of genomic fragments of tropical japonica origin in some indica varieties indicates that refined introgression must have occurred by natural crossbreeding in the course of evolution of the black trait in rice.