Comparative population genomics reveals the domestication history of the peach,Prunus persica,and human influences on perennial fruit crops

Background

Recently, many studies utilizing next generation sequencing have investigated plant evolution and domestication in annual crops. Peach, Prunus persica, is a typical perennial fruit crop that has ornamental and edible varieties. Unlike other fruit crops, cultivated peach includes a large number of phenotypes but few polymorphisms. In this study, we explore the genetic basis of domestication in peach and the influence of humans on its evolution.

Results

We perform large-scale resequencing of 10 wild and 74 cultivated peach varieties, including 9 ornamental, 23 breeding, and 42 landrace lines. We identify 4.6 million SNPs, a large number of which could explain the phenotypic variation in cultivated peach. Population analysis shows a single domestication event, the speciation of P. persica from wild peach. Ornamental and edible peach both belong to P. persica, along with another geographically separated subgroup, Prunus ferganensis.We identify 147 and 262 genes under edible and ornamental selection, respectively. Some of these genes are associated with important biological features. We perform a population heterozygosity analysis in different plants that indicates that free recombination effects could affect domestication history. By applying artificial selection during the domestication of the peach and facilitating its asexual propagation, humans have caused a sharp decline of the heterozygote ratio of SNPs.

Conclusions

Our analyses enhance our knowledge of the domestication history of perennial fruit crops, and the dataset we generated could be useful for future research on comparative population genomics.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0415-1) contains supplementary material, which is available to authorized users.     

Earliest “Domestic” Cats in China Identified as Leopard Cat (Prionailurus bengalensis)

The ancestor of all modern domestic cats is the wildcat, Felis silvestris lybica, with archaeological evidence indicating it was domesticated as early as 10,000 years ago in South-West Asia. A recent study, however, claims that cat domestication also occurred in China some 5,000 years ago and involved the same wildcat ancestor (F. silvestris). The application of geometric morphometric analyses to ancient small felid bones from China dating between 5,500 to 4,900 BP, instead reveal these and other remains to be that of the leopard cat (Prionailurus bengalensis). These data clearly indicate that the origins of a human-cat ‘domestic’ relationship in Neolithic China began independently from South-West Asia and involved a different wild felid species altogether. The leopard cat’s ‘domestic’ status, however, appears to have been short-lived—its apparent subsequent replacement shown by the fact that today all domestic cats in China are genetically related to F. silvestris.     

Archaeological Soybean (Glycine max) in East Asia: Does Size Matter?

The recently acquired archaeological record for soybean from Japan, China and Korea is shedding light on the context in which this important economic plant became associated with people and was domesticated. This paper examines archaeological (charred) soybean seed size variation to determine what insight can be gained from a comprehensive comparison of 949 specimens from 22 sites. Seed length alone appears to represent seed size change through time, although the length × width × thickness product has the potential to provide better size change resolution. A widespread early association of small seeded soybean is as old as 9000-8600 cal BP in northern China and 7000 cal BP in Japan. Direct AMS radiocarbon dates on charred soybean seeds indicate selection resulted in large seed sizes in Japan by 5000 cal BP (Middle Jomon) and in Korea by 3000 cal BP (Early Mumun). Soybean seeds recovered in China from the Shang through Han periods are similar in length to the large Korean and Japanese specimens, but the overall size of the large Middle and Late Jomon, Early Mumun through Three Kingdom seeds is significantly larger than any of the Chinese specimens. The archaeological record appears to disconfirm the hypothesis of a single domestication of soybean and supports the view informed by recent phyologenetic research that soybean was domesticated in several locations in East Asia.     

水稻驯化与长江文明

水稻(即亚洲栽培稻Oryza sativa)是世界上最重要的粮食作物之一, 全球有超过半数以上人口以稻米为食。关于水稻是何时、何地、在什么环境下开始驯化等问题一直是学术界关注的热点。得益于分析技术的进步, 近年来考古学和遗传学研究在水稻驯化起源问题上取得了重要进展。本文简要综述了有关长江流域的水稻驯化起源的遗传学和考古学的研究进展, 并讨论了水稻驯化与稻作文化及长江文明的关系。遗传学研究结果认为水稻(粳稻)最早起源于中国长江流域及以南地区(珠江流域), 考古学证据则表明水稻最先于10,000-8,000 BP在中国长江流域被驯化, 水稻驯化和稻作农业的发展催生了长江文明。这些进展促进了我们对水稻驯化、稻作文化和长江文明的认识, 对长江流域重要植物资源的保护也有启示意义。     

The genetic diversity and population structure of wild soybean evaluated by chloroplast and nuclear gene sequences

Glycine soja, also called wild soybean, is the wild ancestor of domesticated soybean (Glycine max), and one of the world's major cultivated crops. Wild soybean is a valuable resource for the breeding of cultivated soybean and harbors useful genes or agronomic traits. To use and conserve this valuable resource, we conducted a study to evaluate the genetic diversity and population structure of wild soybean using the sequencing data of two nuclear loci (AF105221 and PhyB) and one chloroplast locus (trnQ-rps16) of more than 600 individuals representing 53 populations throughout the natural distribution range. The results showed that most of the variation was found within the populations and groups, but significant genetic differentiation was also detected among different eco-geographical groups. Correlations between genetic and geographical distance at all the loci were consistent with the isolation by distance gene flow model. G. soja exhibited the highest genetic diversity in middle and downstream of Yangzi River (MDYR) region, followed by North East China (NEC), and was the lowest in North West China (NWC). We concluded that both in situ and ex situ conservation strategies required for wild soybean populations, especially which are native to MDYR and NEC regions.     

Early Mixed Farming of Millet and Rice 7800 Years Ago in the Middle Yellow River Region,China

The Peiligang Culture (9000-7000 cal. yr BP) in the Middle Yellow River region, North China, has long been considered representative of millet farming. It is still unclear, however, if broomcorn millet or foxtail millet was the first species domesticated during the Peiligang Culture. Furthermore, it is also unknown whether millet was cultivated singly or together with rice at the same period. In this study, phytolith analysis of samples from the Tanghu archaeological site reveals early crop information in the Middle Yellow River region, China. Our results show that broomcorn millet was the early dry farming species in the Peiligang Culture at 7800 cal. yr BP, while rice cultivation took place from 7800 to 4500 cal. yr BP. Our data provide new evidence of broomcorn millet and rice mixed farming at 7800 cal. yr BP in the Middle Yellow River region, which has implications for understanding the domestication process of the two crops, and the formation and continuance of the Ancient Yellow River Civilization.     

Olive tree varieties cultivated for the great Baetican oil trade between the 1st and the 4th centuries <Emphasis Type="SmallCaps">ad</Emphasis>: morphometric analysis of olive stones from Las Delicias (Ecija,Province of Seville,Spain)

During the excavations of a Roman amphora workshop and oil mill of the 1st–4th century ad in Las Delicias, Genil valley, Ecija, Spain, large quantities of charred olive stones were recovered. The assemblages discovered in the pottery kilns demonstrate the use as fuel of olive residues, which were obtained from the extraction of the oil in the nearby mill. The abundance of material offered the opportunity to study the infra-specific diversity of the olives growing in the province of Baetica, which is known to have been an important oil-producing region during the Roman Empire. In total, 335 intact charred archaeological olive stones were analysed using geometric morphometry (outline analysis) and compared with several current morphotypes. These have been identified within a set of dimensional references of the stones established from the morphometric study of current varieties and wild populations, including genuinely wild and feral forms of olives, from various areas around the Mediterranean. The morphotype mainly found in wild populations was widely represented among the olive stones from Las Delicias. A large proportion of the archaeological stones were however close to various domesticated forms, which reflect the history of the region and of its varied cultural Mediterranean influences, Punic, Greek and Roman. Moreover, intermediate forms between two distinct morphotypes were identified. They suggest that hybrid olive trees derived from crosses among domesticated varieties and also between domesticated and wild forms, were grown in Las Delicias. In the Genil valley, Roman olive cultivation was based on a set of local olives which included wild and domesticated varieties from various origins, and whose diversity arose from breeding for improvement of varieties.     

Molecular Evidence and the Origin and Development of the Domesticated Sunflower (<Emphasis Type="Italic">Helianthus annum</Emphasis>, Asteraceae)

The domesticated sunflower,Helianthus annuus, is an important economic crop, yet molecular data regarding its evolution are limited. Here we review morphological, geographical, archaeological, and molecular evidence pertaining to its origin and development. New isozyme and chloroplast DNA (cpDNA) evidence is also presented.Morphological, geographical, and archaeological evidence has led to the hypothesis that the domesticated sunflower was derived from a wild/weedy form ofH. annuus possibly in the Midwest. Molecular evidence was concordant with this hypothesis. A high degree of enzymatic and cpDNA sequence similarity was observed between wild and domesticatedH. annuus, and domesticatedH. annuus contained a subset of the alleles and cpDNAs found in wildH. annuus. The extensive polymorphism in the wild plants and the virtual monomorphism in cultivated lines for both isozyme and cpDNA phenotypes further suggest a single origin of the domesticated sunflower from a very limited gene pool. In addition, Native American varieties of the domesticated sunflower were genetically more variable than other cultivated lines, possibly indicating that they gave rise to the other cultivated stocks. Molecular evidence did not, however, allow conclusions as to the exact geographic origin of the domesticated sunflower.     

A draft genome of sweet cherry (Prunus avium L.) reveals genome‐wide and local effects of domestication

Sweet cherry (Prunus avium L.) trees are both economically important fruit crops but also important components of natural forest ecosystems in Europe, Asia and Africa. Wild and domesticated trees currently coexist in the same geographic areas with important questions arising on their historical relationships. Little is known about the effects of the domestication process on the evolution of the sweet cherry genome. We assembled and annotated the genome of the cultivated variety “Big Star*” and assessed the genetic diversity among 97 sweet cherry accessions representing three different stages in the domestication and breeding process (wild trees, landraces and modern varieties). The genetic diversity analysis revealed significant genome‐wide losses of variation among the three stages and supports a clear distinction between wild and domesticated trees, with only limited gene flow being detected between wild trees and domesticated landraces. We identified 11 domestication sweeps and five breeding sweeps covering, respectively, 11.0 and 2.4 Mb of the P. avium genome. A considerable fraction of the domestication sweeps overlaps with those detected in the related species, Prunus persica (peach), indicating that artificial selection during domestication may have acted independently on the same regions and genes in the two species. We detected 104 candidate genes in sweep regions involved in different processes, such as the determination of fruit texture, the regulation of flowering and fruit ripening and the resistance to pathogens. The signatures of selection identified will enable future evolutionary studies and provide a valuable resource for genetic improvement and conservation programs in sweet cherry.     

High-throughput sequencing of <Emphasis Type="Italic">Prunus ferganensis</Emphasis> indicates that it is a geographical population of <Emphasis Type="Italic">P. persica</Emphasis>

Peach belongs to the genus Prunus, which includes Prunus persica and its relative species, P. mira, P. davidiana, P. kansuensis, and P. ferganensis. Of these, P. ferganensis have been classified as a species, subspecies, or geographical population of P. persica. To explore the genetic difference between P. ferganensis and P. persica, high-throughput sequencing was used in different peach accessions belonging to different species. First, low-depth sequencing data of peach accessions belonging to four categories revealed that similarity between P. ferganensis and P. persica was similar to that between P. persica accessions from different geographical populations. Then, to further detect the genomic variation in P. ferganensis, the P. ferganensis accession “Xinjiang Pan Tao 1” and the P. persica accession “Xia Miao 1” were sequenced with high depth, and sequence reads were assembled. The results showed that the collinearity of “Xinjiang Pan Tao 1” with the reference genome “Lovell” was higher than that of “Xia Miao 1” and “Lovell” peach. Additionally, the number of genetic variants, including single nucleotide polymorphisms (SNPs), structural variations (SVs), and the specific genes annotated from unmapped sequence in “Xia Miao 1” was higher than that in “Xinjiang Pan Tao 1” peach. The data showed that there was a close distance between “Xinjiang Pan Tao 1” (P. ferganensis) and reference genome which belong to P. persica, comparing “Xia Miao 1” (P. persica) and reference ones. The results accompany with phylogenetic tree and structure analysis confirmed that P. ferganensis should be considered as a geographic population of P. persica rather than a subspecies or a distinct species. Furthermore, gene ontology analysis was performed using the gene comprising large-effect variation to understand the phenotypic difference between two accessions. The result revealed that the pathways of gene function affected by SVs but SNPs and insertion-deletions markedly differed between the two peach accessions.     

全新世早期中国长江下游地区橡子和水稻的开发利用

最近发掘的浙江上山和小黄山遗址(11400—8000cal BP)是长江下游地区迄今发现最早的村落遗址。这两个遗址均出土有大量磨石, 而有机物遗存保存很少。本文对7件石器进行了淀粉粒、植硅体以及矿物残留物的初步分析, 以便检验这些方法在复原古代生计形态和器物功能研究中的可行性。鉴定出的淀粉粒包括橡子、根茎类、薏苡以及疑似菱角等, 但是没有发现水稻。在发现的水稻植硅体中, 来自茎杆和叶的密度较高, 而来自颖壳的密度较低。矿物分析表明残留物来自工具的使用以及周围的土壤基质。结果显示这些磨石曾经用于加工富含淀粉的植物和制陶用的羼和料。我们认为, 对富含淀粉并且适于长期贮存的植物特别是橡子的大量开发利用是全新世早期长江下游地区生计形态中采集策略的特征。本研究显示了淀粉粒和植硅体分析在古代生计重建中的巨大潜力。对这些遗址残留物的进一步研究将有助于我们更好地理解这一地区从采集经济到定居农耕经济的转变。     

Landscape use and fruit cultivation in Petra (Jordan) from Early Nabataean to Byzantine times (2nd century <Emphasis Type="SmallCaps">bc</Emphasis>–5th century <Emphasis Type="SmallCaps">ad</Emphasis>)

Archaeobotanical analyses of charred seeds, fruit and wood charcoal from the residential part of the ez-Zantur area at Petra, Jordan, provide new data on the agricultural economy and use of the landscape in this famous merchant Nabataean city from the middle of the 2nd century bc to the beginning of the 5th century ad. The study is based on analyses of 7,640 whole and fragmented seeds, pips and fruit stones and 624 charcoal fragments sampled from household deposits. The results show that the food supply was based on common Mediterranean cultivated taxa such as cereals (Hordeum vulgare, Triticum aestivum/durum), pulses (Lens culinaris) and fruit (Olea europaea, Ficus carica, Vitis vinifera), which were probably cultivated both in the city and its hinterland. The by-products from the processing of cereals and fruit trees played a significant role in fuel supply, supplementing woody wild plants obtained from rocky slopes and the desert valley. The variety of fuel resources shows a major capacity to manage complex supply networks and perhaps the rarity of natural woodland cover. The existence of orchards within the city centre and notably olive groves is indicated in the Early Nabataean period (mid 2nd century to mid 1st century bc) but they expanded during the Classical Nabataean period (mid 1st century bc to 1st century ad), probably reflecting specialised fruit growing. Unusual plant remains such as Prunus armeniaca (apricot), P. persica (peach) and Juglans regia (walnut) are considered to be social indicators of prosperity. These archaeobotanical results fit with others from this region and match with the urbanization and social dynamics of the city of Petra.     

Crop water availability in early agriculture: evidence from carbon isotope discrimination of seeds from a tenth millennium BP site on the Euphrates

The analysis of carbon isotope discrimination (Δ) in crop plant remains from archaeological sites may help to assess water availability for early agriculture. This study presents the analysis of Δ in seeds of naked wheat (Triticum aestivum/durum), lentil (Lens orientalis/culinaris), and flax (Linum sp.) found at the archaeological site of Tell Halula in the valley of the Middle Euphrates (Syria). This Neolithic site is the oldest in this region of the Fertile Crescent where the cultivation of domesticated plants has been reported, with seed remains ranging from 9550 to 8465 BP. Most of the seeds analysed showed Δ values greater than 16 ‰, reaching 20 ‰ for some samples of flax. For wheat, Δ values were much higher than those reported in present-day (1996) durum wheat crops cultivated under rainfed conditions in north-west Syria under environments with somewhat higher rainfall than Tell Halula. Similarly, grains of present-day (1997) barley cultivated in the archaeological site also showed lower values than those found in archaeological kernels. An empirical relationship between Δ of mature kernels and total precipitation (plus irrigation where applicable) from heading to maturity (r² = 0.82, n = 11) was established for durum wheat, currently cultivated in different environments of the Mediterranean basin. The resulting relationship was applied to the data on Δ of wheat fossil kernels from Tell Halula to estimate the accumulated water inputs during the time (about 6 weeks) the kernels were produced. Calculated water inputs for wheat during early agriculture were (over 110 mm) at least 5 times higher than current-day rainfall accumulated in Tell Halula during the same phenological period. These results strongly suggest that early agriculture wheat was cultivated at Tell Halula under much wetter conditions than are currently to be found in the area. The presence of flax and its very high Δ values also support this conclusion. Whether such humid conditions during cultivation were due to moister conditions prevailing at this time, by planting in alluvial areas or by irrigation works is discussed.     

Development and evaluation of a 9K SNP array for peach by internationally coordinated SNP detection and validation in breeding germplasm

Although a large number of single nucleotide polymorphism (SNP) markers covering the entire genome are needed to enable molecular breeding efforts such as genome wide association studies, fine mapping, genomic selection and marker-assisted selection in peach [Prunus persica (L.) Batsch] and related Prunus species, only a limited number of genetic markers, including simple sequence repeats (SSRs), have been available to date. To address this need, an international consortium (The International Peach SNP Consortium; IPSC) has pursued a coordinated effort to perform genome-scale SNP discovery in peach using next generation sequencing platforms to develop and characterize a high-throughput Illumina Infinium® SNP genotyping array platform. We performed whole genome re-sequencing of 56 peach breeding accessions using the Illumina and Roche/454 sequencing technologies. Polymorphism detection algorithms identified a total of 1,022,354 SNPs. Validation with the Illumina GoldenGate® assay was performed on a subset of the predicted SNPs, verifying ∼75% of genic (exonic and intronic) SNPs, whereas only about a third of intergenic SNPs were verified. Conservative filtering was applied to arrive at a set of 8,144 SNPs that were included on the IPSC peach SNP array v1, distributed over all eight peach chromosomes with an average spacing of 26.7 kb between SNPs. Use of this platform to screen a total of 709 accessions of peach in two separate evaluation panels identified a total of 6,869 (84.3%) polymorphic SNPs.The almost 7,000 SNPs verified as polymorphic through extensive empirical evaluation represent an excellent source of markers for future studies in genetic relatedness, genetic mapping, and dissecting the genetic architecture of complex agricultural traits. The IPSC peach SNP array v1 is commercially available and we expect that it will be used worldwide for genetic studies in peach and related stone fruit and nut species.     

Morphometric Analysis of Sunflower (<Emphasis Type="Italic">Helianthus annuus</Emphasis> L.) Achenes from Mexico and Eastern North America<Superscript>1</Superscript>

Morphometric Analysis of Sunflower ( Helianthus annuus L.) Achenes from Mexico and Eastern North America. Sunflower (Helianthus annuus L.) has played a major role in the evolution of agricultural systems in the Americas. The discovery of ancient domesticated remains from archaeological deposits in pre-Columbian Mexico offers new dimensions to widely accepted viewpoints on the domestication pattern of H. annuus. Although American sunflower populations north of Mexico have been examined extensively, Mexican indigenous domesticated landraces have not been studied in any detail. In this study, we morphologically assessed wild and domesticated sunflower achenes from Mexico and compared them to similar datasets from eastern North America. Additionally, we evaluated the utility of four computer-assisted shape measurements in discriminating between wild and domesticated sunflower achenes (fruits) and compared variation in achene size among modern wild and cultivated populations from both Mexico and the U.S. We found that, of the shape parameters tested, none were informative in distinguishing wild achenes from domesticated varieties. Subsequent size analysis, using conventional parameters of length, width, and thickness, showed that modern wild populations from Mexico had smaller achenes compared to modern populations from eastern North America. Domesticated achenes unearthed from Mexican archaeological sites, however, were significantly larger than the early domesticated specimens recovered from eastern North America. Our methodological results indicate that variation in archaeological sunflower achenes is better described by conventional size parameters rather than computerized shape analysis.     

Genome Analysis Traces Regional Dispersal of Rice in Taiwan and Southeast Asia

The dispersal of rice (Oryza sativa) following domestication influenced massive social and cultural changes across South, East, and Southeast (SE) Asia. The history of dispersal across islands of SE Asia, and the role of Taiwan and the Austronesian expansion in this process remain largely unresolved. Here, we reconstructed the routes of dispersal of O. sativa ssp. japonica rice to Taiwan and the northern Philippines using whole-genome resequencing of indigenous rice landraces coupled with archaeological and paleoclimate data. Our results indicate that japonica rice found in the northern Philippines diverged from Indonesian landraces as early as 3,500 years before present (BP). In contrast, rice cultivated by the indigenous peoples of the Taiwanese mountains has complex origins. It comprises two distinct populations, each best explained as a result of admixture between temperate japonica that presumably came from northeast Asia, and tropical japonica from the northern Philippines and mainland SE Asia, respectively. We find that the temperate japonica component of these indigenous Taiwan populations diverged from northeast Asia subpopulations at about 2,600 BP, whereas gene flow from the northern Philippines had begun before ∼1,300  BP. This coincides with a period of intensified trade established across the South China Sea. Finally, we find evidence for positive selection acting on distinct genomic regions in different rice subpopulations, indicating local adaptation associated with the spread of japonica rice.     

Molecular Evidence and the Origin and Development of the Domesticated Sunflower (Helianthus annum, Asteraceae)

The domesticated sunflower,Helianthus annuus, is an important economic crop, yet molecular data regarding its evolution are limited. Here we review morphological, geographical, archaeological, and molecular evidence pertaining to its origin and development. New isozyme and chloroplast DNA (cpDNA) evidence is also presented. Morphological, geographical, and archaeological evidence has led to the hypothesis that the domesticated sunflower was derived from a wild/weedy form ofH. annuus possibly in the Midwest. Molecular evidence was concordant with this hypothesis. A high degree of enzymatic and cpDNA sequence similarity was observed between wild and domesticatedH. annuus, and domesticatedH. annuus contained a subset of the alleles and cpDNAs found in wildH. annuus. The extensive polymorphism in the wild plants and the virtual monomorphism in cultivated lines for both isozyme and cpDNA phenotypes further suggest a single origin of the domesticated sunflower from a very limited gene pool. In addition, Native American varieties of the domesticated sunflower were genetically more variable than other cultivated lines, possibly indicating that they gave rise to the other cultivated stocks. Molecular evidence did not, however, allow conclusions as to the exact geographic origin of the domesticated sunflower.     

A major locus for resistance to <Emphasis Type="Italic">Botryosphaeria dothidea</Emphasis> in <Emphasis Type="Italic">Prunus</Emphasis>

Species in the fungal family Botryosphaeriaceae are significant pathogens of peach. The climatic conditions in the Southeastern USA are conducive to the development of peach fungal gummosis (PFG) with an estimated yield reduction of up to 40% in severe cases. Genotypes with resistance to this PFG were identified in interspecific crosses and segregating backcross populations generated using Kansu peach (Prunus kansuensis Rehder), almond [Prunus dulcis (Mill.) D.A. Webb], and peach [Prunus persica (L.) Batsch]. Hybrids were evaluated for four consecutive years in field conditions. Data generated was validated in different environments using clonal replicates of the hybrids. The F₁ and BC₁F₁ segregation population data suggest a dominant allele for PFG resistance originating from almond. Segregation and mapping analysis located the PFG resistance locus on a chimeric linkage groups 6–8 near the leaf color locus. The molecular markers identified will facilitate marker-assisted selection (MAS) and introgression of this resistance trait into commercial peach germplasm.     

Bioinformatics prediction of miRNAs in the Prunus persica genome with validation of their precise sequences by miR-RACE

We predicted 262 potential MicroRNAs (miRNAs) belonging to 70 miRNA families from the peach (Prunus persica) genome and two specific 5′ and 3′ miRNA rapid amplification of cDNA ends (miR-RACE) PCR reactions and sequence-directed cloning were employed to accurately validate 61 unique P. persica miRNAs (Ppe-miRNAs) sequences belonging to 61 families comprising 97 Ppe-miRNAs. Validation of the termini nucleotides in particular can define the real sequences of the Ppe-miRNAs on peach genome. Comparison between predicted and validated Ppe-miRNAs through alignment revealed that 43 unique orthologous sequences were identical, while the remaining 18 exhibited some divergences at their termini nucleotides. Quantitative real-time polymerase chain reaction (qRT-PCR) was further employed to analyze the expression of all the 61 miRNAs and 10 putative targets of 8 randomly selected Ppe-miRNAs in peach leaves, flowers and fruits at different stages of development, where both the miRNAs and the putative target genes showed tissue-specific expression.