Genome assembly and association tests identify interacting loci associated with vigor, precocity, and sex in interspecific pistachio rootstocks.

Understanding the basis of hybrid vigor remains a key question in crop breeding and improvement, especially for rootstock development where F1 hybrids are extensively utilized. Full-sibling UCB-1 F1 seedling rootstocks are widely planted in commercial pistachio orchards that are generated by crossing 2 highly heterozygous outbreeding parental trees of Pistacia atlantica (female) and P. integerrima (male). This results in extensive phenotypic variability, prompting costly removal of low-yielding small trees. To identify the genetic basis of this variability, we assembled chromosome-scale genome assemblies of the parental trees of UCB-1. We genotyped 960 UCB-1 trees in an experimental orchard for which we also collected multiyear phenotypes. We genotyped an additional 1,358 rootstocks in 6 commercial pistachio orchards and collected single-year tree-size data. Genome-wide single marker association tests identified loci associated with tree size and shape, sex, and precocity. In the experimental orchard, we identified multiple trait-associated loci and a strong candidate for ZZ/ZW sex chromosomes. We found significant marker associations unique to different traits and to early vs late phenotypic measures of the same trait. We detected 2 loci strongly associated with rootstock size in commercial orchards. Pseudo-testcross classification of markers demonstrated that the trait-associated alleles for each locus were segregating in the gametes of opposite parents. These 2 loci interact epistatically to generate the bimodal distribution of tree size with undesirable small trees observed by growers. We identified candidate genes within these regions. These findings provide a foundational resource for marker development and genetic selection of vigorous pistachio UCB-1 rootstock.

Pistachio genomes provide insights into nut tree domestication and ZW sex chromosome evolution.

Pistachio is a nut crop domesticated in the Fertile Crescent and a dioecious species with ZW sex chromosomes. We sequenced the genomes of Pistacia vera cultivar (cv.) Siirt, the female parent, and P. vera cv. Bagyolu, the male parent. Two chromosome-level reference genomes of pistachio were generated, and Z and W chromosomes were assembled. The ZW chromosomes originated from an autosome following the first inversion, which occurred approximately 8.18 Mya. Three inversion events in the W chromosome led to the formation of a 12.7-Mb (22.8% of the W chromosome) non-recombining region. These W-specific sequences contain several genes of interest that may have played a pivotal role in sex determination and contributed to the initiation and evolution of a ZW sex chromosome system in pistachio. The W-specific genes, including defA, defA-like, DYT1, two PTEN1, and two tandem duplications of six VPS13A paralogs, are strong candidates for sex determination or differentiation. Demographic history analysis of resequenced genomes suggest that cultivated pistachio underwent severe domestication bottlenecks approximately 7640 years ago, dating the domestication event close to the archeological record of pistachio domestication in Iran. We identified 390, 211, and 290 potential selective sweeps in 3 cultivar subgroups that underlie agronomic traits such as nut development and quality, grafting success, flowering time shift, and drought tolerance. These findings have improved our understanding of the genomic basis of sex determination/differentiation and horticulturally important traits and will accelerate the improvement of pistachio cultivars and rootstocks.

Sugar, Invertase Enzyme Activities and Invertase Gene Expression in Different Developmental Stages of Strawberry Fruits.

The cultivated strawberry (Fragaria × ananassa) is octoploid (2n = 8x = 56) and has been the focused fruit species of which an increasing number of molecular and genetic research has been conducted in recent years. The aim of this study is to identify the relationships between sucrose metabolism, invertase enzyme activity and gene expression in four different fruit development periods (red, pink, green and white) of two commercially important strawberry varieties 'Rubygem' and 'Fortuna'. The metabolite profiles (glucose, fructose, sucrose and total sugar content) of two varieties were discovered to be extremely similar. The highest amount of total sugar was found in red fruits, while the lowest was obtained from green fruits. Invertase represents one of the key enzymes in sucrose metabolism. The lowest invertase activity was obtained from the green fruits in 'Rubygem' and 'Fortuna' during four developmental periods. In these varieties, the amount of sucrose was found to be close to glucose and fructose and the lowest amount was detected in green period, while invertase activity was relatively high during red and pink periods and invertase gene expression was determined at high levels in both primers (St-4 and St-6) in the green period. The results of the study indicated that sugar content and invertase activity were positively correlated while enzyme activity and gene expression were negatively correlated.

Nutritional Analysis of Red-Purple and White-Fleshed Pitaya (Hylocereus) Species.

Pitaya is one of the most preferred and produced tropical fruit species recently introduced to the Mediterrranean region in Turkey. Due to its nutritional fruits with high economic value, the popularity of pitaya increases steadily in Turkey as an alternative crop. No detailed nutritional analysis has been undertaken in Turkey so far on fruits of the pitaya species. In this study, we determined and compared some nutritional parameters in fruit flesh of two pitaya (dragon fruit) species (Hylocereus polyrhizus: Siyam and Hylocereus undatus: Vietnam Jaina) grown in the Adana province located in the eastern Mediterranean region in Turkey. The individual sugars, antioxidant activity, total phenolic content, phenolic compounds and volatiles were determined for the first time in Turkey on two pitaya species. The results showed that total phenol content and antioxidant capacity are notably higher in red-fleshed fruits than white-fleshed ones and the predominant phenolic compound in fruits of both species was quercetin. The total sugar content and most of the phenolic compounds in fruits of two pitaya species were similar. A total of 51 volatile compounds were detected by using two Solid Phase Micro Extraction (SPME) fibers, coupled with Gas Chromatography Mass Spectrometry (GC-MS) techniques, and more volatile compounds were presented in the white-fleshed species. Total phenolic content (TPC) of the red-fleshed and white-fleshed pitaya species were 16.66 and 17.11 mg GAE/100 g FW (fresh weight). This study provides a first look at the biochemical comparison of red-fleshed and white-fleshed pitaya species introduced and cultivated in Turkey. The results also showed, for the first time, the biochemical content and the potential health benefit of Hylocereus grown in different agroecological conditions, providing important information for pitaya researchers and application perspective.

Advances in Rootstock Breeding of Nut Trees: Objectives and Strategies.

The production and consumption of nuts are increasing in the world due to strong economic returns and the nutritional value of their products. With the increasing role and importance given to nuts (i.e., walnuts, hazelnut, pistachio, pecan, almond) in a balanced and healthy diet and their benefits to human health, breeding of the nuts species has also been stepped up. Most recent fruit breeding programs have focused on scion genetic improvement. However, the use of locally adapted grafted rootstocks also enhanced the productivity and quality of tree fruit crops. Grafting is an ancient horticultural practice used in nut crops to manipulate scion phenotype and productivity and overcome biotic and abiotic stresses. There are complex rootstock breeding objectives and physiological and molecular aspects of rootstock-scion interactions in nut crops. In this review, we provide an overview of these, considering the mechanisms involved in nutrient and water uptake, regulation of phytohormones, and rootstock influences on the scion molecular processes, including long-distance gene silencing and trans-grafting. Understanding the mechanisms resulting from rootstock × scion × environmental interactions will contribute to developing new rootstocks with resilience in the face of climate change, but also of the multitude of diseases and pests.

Transcriptome Sequencing and Development of Novel Genic SSR Markers From Pistacia vera L.

In this study, we aimed to develop novel genic simple sequence repeat (eSSR) markers and to study phylogenetic relationship among Pistacia species. Transcriptome sequencing was performed in different tissues of Siirt and Atl cultivars of pistachio (Pistacia vera). A total of 37.5-Gb data were used in the assembly. The number of total contigs and unigenes was calculated as 98,831, and the length of N50 was 1,333 bp after assembly. A total of 14,308 dinucleotide, trinucleotide, tetranucleotide, pentanucleotide, and hexanucleotide SSR motifs (4-17) were detected, and the most abundant SSR repeat types were trinucleotide (29.54%), dinucleotide (24.06%), hexanucleotide (20.67%), pentanucleotide (18.88%), and tetranucleotide (6.85%), respectively. Overall 250 primer pairs were designed randomly and tested in eight Pistacia species for amplification. Of them, 233 were generated polymerase chain reaction products in at least one of the Pistacia species. A total of 55 primer pairs that had amplifications in all tested Pistacia species were used to characterize 11 P. vera cultivars and 78 wild Pistacia genotypes belonging to nine Pistacia species (P. khinjuk, P. eurycarpa, P. atlantica, P. mutica, P. integerrima, P. chinensis, P. terebinthus, P. palaestina, and P. lentiscus). A total of 434 alleles were generated from 55 polymorphic eSSR loci with an average of 7.89 alleles per locus. The mean number of effective allele was 3.40 per locus. Polymorphism information content was 0.61, whereas observed (Ho) and expected heterozygosity (He) values were 0.39 and 0.65, respectively. UPGMA (unweighted pair-group method with arithmetic averages) and STRUCTURE analysis divided 89 Pistacia genotypes into seven populations. The closest species to P. vera was P. khinjuk. P. eurycarpa was closer P. atlantica than P. khinjuk. P. atlantica-P. mutica and P. terebinthus-P. palaestina pairs of species were not clearly separated from each other, and they were suggested as the same species. The present study demonstrated that eSSR markers can be used in the characterization and phylogenetic analysis of Pistacia species and cultivars, as well as genetic linkage mapping and QTL (quantitative trait locus) analysis.

Major QTL with pleiotropic effects controlling time of leaf budburst and flowering-related traits in walnut (Juglans regia L.).

Breeding studies in walnut (Juglans regia L.) are usually time consuming due to the long juvenile period and therefore, this study aimed to determine markers associated with time of leaf budburst and flowering-related traits by performing a genome-wide association study (GWAS). We investigated genotypic variation and its association with time of leaf budburst and flowering-related traits in 188 walnut accessions. Phenotypic data was obtained from 13 different traits during 3 consecutive years. We used DArT-seq for genotyping with a total of 33,519 (14,761 SNP and 18,758 DArT) markers for genome-wide associations to identify marker underlying these traits. Significant correlations were determined among the 13 different traits. Linkage disequilibrium decayed very quickly in walnut in comparison with other plants. Sixteen quantitative trait loci (QTL) with major effects (R2 between 0.08 and 0.23) were found to be associated with a minimum of two phenotypic traits each. Of these QTL, QTL05 had the maximum number of associated traits (seven). Our study is GWAS for time of leaf budburst and flowering-related traits in Juglans regia L. and has a strong potential to efficiently implement the identified QTL in walnut breeding programs.

Characterization of hawthorn (Crataegus spp.) genotypes by SSR markers.

Hawthorn (Crataegus spp.) is an edible wild fruit that is used in traditional medicine, landscape studies, and food and beverage industries in many countries. It is an important wild plant species in Turkey and is numerous in the Yozgat Province. Genetic and breeding studies on hawthorn are very limited. Therefore, we aimed to characterize 91 hawthorn genotypes using simple sequence repeat (SSR) markers. The SSRs were developed from apple and pear and were screened in hawthorn for amplification and polymorphisms. A total of 265 alleles were detected from thirty-two SSR primer pairs, and those were used to identify genetic relationships. The number of alleles ranged from 2 to 21 alleles per locus with a mean value of 8.28. The Hi05b09 locus showed the highest allele number (Na = 21). The polymorphism information content (PIC) values ranged from 0.16 (CH03d10) to 0.89 (C6554) with a mean value of 0.60. An Unweighted Pair Group Method with Arithmetic Average method was used to cluster the genotypes, and four major clusters were obtained from the amplification of the SSRs. STRUCTURE software identified four populations (ΔK = 4) and eight sub-populations (ΔK = 8), and four major clusters similar results to UPGMA analysis. Our study showed that the SSR markers could be utilized as a reliable tool for the determination of genetic variations and relationships of hawthorn genotypes. A basic molecular analysis on the hawthorn genotypes identified in this study will promote the collection of germplasm collection and the selection of parents' in future cross-breeding studies.

Evaluation of Some Phenological and Biochemical Characteristics of Selected New Late Flowering Dried Apricot Cultivars.

In this study, promising candidates of six apricot genotypes were compared with our local and major cultivar (Hacihaliloglu) based on their phenological, pomological, and biochemical characteristics. Fruit weight, stone weight, flesh firmness, flesh fruit/stone ratio, TSS (Total soluble solids), glucose, fructose, sucrose, total phenols, total antioxidant activity (FRAP and ABTS assays) and bud break, first bloom, full bloom, and harvesting date of apricot cultivar candidates were compared with cultivar Hacihaliloglu 'HH.' According to the obtained results, the higher content of total phenolic compounds and total antioxidant activity was determined in the N95 genotype (96.87 µmol TE/g) fruit, whereas the 'HH' cultivar was characterized by the lower value (94.6 µmol TE/g) especially determined by FRAP method. Individual sugars and the soluble solids content of fruit differed between selected genotypes and 'HH' cultivar. Glucose, fructose, and sucrose contents were higher in all selected genotypes than in Hacihaliloglu cultivar.

Genome survey of pistachio (Pistacia vera L.) by next generation sequencing: Development of novel SSR markers and genetic diversity in Pistacia species.

BACKGROUND: Pistachio (Pistacia vera L.) is one of the most important nut crops in the world. There are about 11 wild species in the genus Pistacia, and they have importance as rootstock seed sources for cultivated P. vera and forest trees. Published information on the pistachio genome is limited. Therefore, a genome survey is necessary to obtain knowledge on the genome structure of pistachio by next generation sequencing. Simple sequence repeat (SSR) markers are useful tools for germplasm characterization, genetic diversity analysis, and genetic linkage mapping, and may help to elucidate genetic relationships among pistachio cultivars and species. RESULTS: To explore the genome structure of pistachio, a genome survey was performed using the Illumina platform at approximately 40× coverage depth in the P. vera cv. Siirt. The K-mer analysis indicated that pistachio has a genome that is about 600 Mb in size and is highly heterozygous. The assembly of 26.77 Gb Illumina data produced 27,069 scaffolds at N50 = 3.4 kb with a total of 513.5 Mb. A total of 59,280 SSR motifs were detected with a frequency of 8.67 kb. A total of 206 SSRs were used to characterize 24 P. vera cultivars and 20 wild Pistacia genotypes (four genotypes from each five wild Pistacia species) belonging to P. atlantica, P. integerrima, P. chinenesis, P. terebinthus, and P. lentiscus genotypes. Overall 135 SSR loci amplified in all 44 cultivars and genotypes, 41 were polymorphic in six Pistacia species. The novel SSR loci developed from cultivated pistachio were highly transferable to wild Pistacia species. CONCLUSIONS: The results from a genome survey of pistachio suggest that the genome size of pistachio is about 600 Mb with a high heterozygosity rate. This information will help to design whole genome sequencing strategies for pistachio. The newly developed novel polymorphic SSRs in this study may help germplasm characterization, genetic diversity, and genetic linkage mapping studies in the genus Pistacia.

Identification of sex-linked SNP markers using RAD sequencing suggests ZW/ZZ sex determination in Pistacia vera L.

BACKGROUND: Pistachio (Pistacia vera L.) is a dioecious species that has a long juvenility period. Therefore, development of marker-assisted selection (MAS) techniques would greatly facilitate pistachio cultivar-breeding programs. The sex determination mechanism is presently unknown in pistachio. The generation of sex-linked markers is likely to reduce time, labor, and costs associated with breeding programs, and will help to clarify the sex determination system in pistachio. RESULTS: Restriction site-associated DNA (RAD) markers were used to identify sex-linked markers and to elucidate the sex determination system in pistachio. Eight male and eight female F1 progenies from a Pistacia vera L. Siirt × Bagyolu cross, along with the parents, were subjected to RAD sequencing in two lanes of a Hi-Seq 2000 sequencing platform. This generated 449 million reads, comprising approximately 37.7 Gb of sequences. There were 33,757 polymorphic single nucleotide polymorphism (SNP) loci between the parents. Thirty-eight of these, from 28 RAD reads, were detected as putative sex-associated loci in pistachio. Validation was performed by SNaPshot analysis in 42 mature F1 progenies and in 124 cultivars and genotypes in a germplasm collection. Eight loci could distinguish sex with 100% accuracy in pistachio. To ascertain cost-effective application of markers in a breeding program, high-resolution melting (HRM) analysis was performed; four markers were found to perfectly separate sexes in pistachio. Because of the female heterogamety in all candidate SNP loci, we report for the first time that pistachio has a ZZ/ZW sex determination system. As the reported female-to-male segregation ratio is 1:1 in all known segregating populations and there is no previous report of super-female genotypes or female heteromorphic chromosomes in pistachio, it appears that the WW genotype is not viable. CONCLUSION: Sex-linked SNP markers were identified and validated in a large germplasm and proved their suitability for MAS in pistachio. HRM analysis successfully validated the sex-linked markers for MAS. For the first time in dioecious pistachio, a female heterogamety ZW/ZZ sex determination system is suggested.

Pistillate flower development and pollen tube growth mode during the delayed fertilization stage in Corylus heterophylla Fisch.

Unlike most angiosperms, in which fertilization occurs within several days after pollination, fertilization in hazel (Corylus Spp.) is delayed by two to three and a half months. However, the female inflorescences or young fruits are too hard or lignified to be dissected according to regular paraffin sectioning technique. So, what the nature of development during the extended progamic phases of hazel remains unknown. The female inflorescence development and pollen tube growth mode during the delayed fertilization stage in hazel were investigated by improved paraffin sectioning and aniline blue staining of pollen tubes. The results showed ovaries and ovules of hazel were invisible at the time of blooming. Early ovary and ovule primordium began to form from 15 to 20 days after blooming, respectively. Integument and mature embryo sacs differentiated from the nucellus on 40th and 55th day after blooming, respectively. Pollen tubes were retarded in the bottom of the style or the pollen tube cavity (PTC, a specifical lignified cavity structure at the bottom of style for pollen tube to rest during progamic phase) for about 26 days. Then, the pollen tubes were observed to leave the PTC and began to enter the ovary. After that, a single pollen tube passed through the vicinity of the micropyle. Finally, pollen tubes turned a corner and penetrated the embryo sac through the tissue of the chalaza instead of micropyle on 52 and 55 days after blooming, respectively. The results of more in-depth information will be beneficial to better understanding of the delayed fertilization process in hazel.

Molecular characterization of mulberry (Morus spp.) genotypes via RAPD and ISSR.

BACKGROUND: In recent years, DNA-based markers have been used quite extensively because of their many advantages over the traditional morphological and biochemical markers. Many studies have shown that molecular markers are useful in delineating the genetic relationships among closely related mulberry genotypes and cultivars. Thus, in the present study, polymer chain reaction based DNA fingerprinting techniques were used to investigate the genetic relationships among mulberry genotypes growing in different agro-climatic regions of Turkey. RESULTS: 20 RAPD primers generated a total of 173 bands, of which 157 (90.75%) were polymorphic. As for 11 ISSR primers, 124 bands (96.55%) were polymorphic in a total of 128. The similarity index for RAPD technique ranged between 0.24-0.98; 25Is203 with 25Is112 were found to be the closest genotypes, while 24Ke10 and 25Is123 were the most distant ones. According to the ISSR result, the genetic similarity index changed between 0.21-095; 25Is203 with 25Is112 genotypes were the closest, while 25Is08 and 01KaD2 were the most distant ones. CONCLUSION: The RAPD and ISSR markers were found to be promising for assessing genetic diversity in mulberry genotypes.

Genetic characterization of pomegranate (Punica granatum L.) genotypes by AFLP markers.

The Coruh Valley, located in Northeastern Turkey, is one of the most important centers of diversity in pomegranate in Turkey. In this study, we attempted to characterize 19 promising pomegranate genotypes originating from the Coruh Valley in using fluorescent dye AFLP markers and capillary electrophoresis. Four AFLP primer combinations were used, generating a total of 297 fragments, 213 of which were polymorphic (73.0%). Resolving powers of the AFLP primers ranged from 0.700 to 1.018, with a total of 3.440, while polymorphism information contents ranged from 0.707 to 0.837 with an average of 0.764. UPGMA clustering of the genotypes showed two major groups. Most of the fruit characteristics of the genotypes within the same group were variable. Therefore, the results showed that molecular characterization is necessary to get reliable relationships among pomegranate genotypes and AFLP markers can be used effectively in pomegranate.

Genetic characterization of pomegranate (Punica granatum L.) genotypes by AFLP markers

The Coruh Valley, located in Northeastern Turkey, is one of the most important centers of diversity in pomegranate in Turkey. In this study, we attempted to characterize 19 promising pomegranate genotypes originating from the Coruh Valley in using fluorescent dye AFLP markers and capillary electrophoresis. Four AFLP primer combinations were used, generating a total of 297 fragments, 213 of which were polymorphic (73.0%). Resolving powers of the AFLP primers ranged from 0.700 to 1.018, with a total of 3.440, while polymorphism information contents ranged from 0.707 to 0.837 with an average of 0.764. UPGMA clustering of the genotypes showed two major groups. Most of the fruit characteristics of the genotypes within the same group were variable. Therefore, the results showed that molecular characterization is necessary to get reliable relationships among pomegranate genotypes and AFLP markers can be used effectively in pomegranate.

Genetic relationships among South-East Turkey wild barley populations and sampling strategies of Hordeum spontaneum.

To assess the genetic diversity and the genetic structure of Turkish wild barley (Hordeum spontaneum Tell.) populations, 76 genotypes from ten ecologically and geographically different locations were analyzed by means of amplified fragment length polymorphism (AFLP) markers. Five primer combinations produced 187 scorable bands, of which 117 (62.6%) were polymorphic. Several population-specific and genotype-specific bands were identified, which differentiate populations or genotypes. Genetic distance, determined by Nei's distance coefficient, varied from 0.07 to 0.21 with an average of 0.13. In the UPGMA dendrogram based on Nei genetic distances, the Hordeum spontaneum populations were separated into two major clusters. Genetic diversity was larger among (68%) than within (32%) populations. Eight AFLP bands were strongly correlated to the altitude of the collecting site, while no clear trend was detected between geographical origin and genetic diversity. Our results strongly suggest the need for a change in Hordeum spontaneum sampling strategy: more populations, rather then more individuals within population, should be sampled to appraise and safeguard genetic diversity in the wild barley gene pool.