Noninfectious Bud-failure Research Articles

Advances in the understanding of noninfectious bud failure in almond

Advances in the understanding of noninfectious bud failure in almond

A review of plant epigenetics through the lens of almond.

While genomes were originally seen as static entities that stably held and organized genetic information, recent advances in sequencing have uncovered the dynamic nature of the genome. New conceptualizations of the genome include complex relationships between the environment and gene expression that must be maintained, regulated, and sometimes even transmitted over generations. The discovery of epigenetic mechanisms has allowed researchers to understand how traits like phenology, plasticity, and fitness can be altered without changing the underlying deoxyribonucleic acid sequence. While many discoveries were first made in animal systems, plants provide a particularly complex set of epigenetic mechanisms due to unique aspects of their biology and interactions with human selective breeding and cultivation. In the plant kingdom, annual plants have received the most attention; however, perennial plants endure and respond to their environment and human management in distinct ways. Perennials include crops such as almond, for which epigenetic effects have long been linked to phenomena and even considered relevant for breeding. Recent discoveries have elucidated epigenetic phenomena that influence traits such as dormancy and self-compatibility, as well as disorders like noninfectious bud failure, which are known to be triggered by the environment and influenced by inherent aspects of the plant. Thus, epigenetics represents fertile ground to further understand almond biology and production and optimize its breeding. Here, we provide our current understanding of epigenetic regulation in plants and use almond as an example of how advances in epigenetics research can be used to understand biological fitness and agricultural performance in crop plants.

Whole-genome sequence and methylome profiling of the almond [Prunus dulcis (Mill.) D.A. Webb] cultivar 'Nonpareil'.

Almond [Prunus dulcis (Mill.) D.A. Webb] is an economically important, specialty nut crop grown almost exclusively in the United States. Breeding and improvement efforts worldwide have led to the development of key, productive cultivars, including ‘Nonpareil,’ which is the most widely grown almond cultivar. Thus far, genomic resources for this species have been limited, and a whole-genome assembly for ‘Nonpareil’ is not currently available despite its economic importance and use in almond breeding worldwide. We generated a 571X coverage genome sequence using Illumina, PacBio, and optical mapping technologies. Gene prediction revealed 49,321 putative genes using MinION Oxford nanopore and Illumina RNA sequencing, and genome annotation found that 68% of predicted models are associated with at least one biological function. Furthermore, epigenetic signatures of almond, namely DNA cytosine methylation, have been implicated in a variety of phenotypes including self-compatibility, bud dormancy, and development of noninfectious bud failure. In addition to the genome sequence and annotation, this report also provides the complete methylome of several almond tissues, including leaf, flower, endocarp, mesocarp, exocarp, and seed coat. Comparisons between methylation profiles in these tissues revealed differences in genome-wide weighted % methylation and chromosome-level methylation enrichment.

Identification of Putative Markers of Non-infectious Bud Failure in Almond [Prunus dulcis (Mill.) D.A. Webb] Through Genome Wide DNA Methylation Profiling and Gene Expression Analysis in an Almond × Peach Hybrid Population.

Almond [Prunus dulcis (Mill.) D.A. Webb] is an economically important nut crop susceptible to the genetic disorder, Non-infectious Bud Failure (NBF). Despite the severity of exhibition in several prominent almond cultivars, no causal mechanism has been identified underlying NBF development. The disorder is hypothesized to be associated with differential DNA methylation patterns based on patterns of inheritance (i.e., via sexual reproduction and clonal propagation) and previous work profiling methylation in affected trees. Peach (Prunus persica L. Batsch) is a closely related species that readily hybridizes with almond; however, peach is not known to exhibit NBF. A cross between an NBF-exhibiting ‘Carmel’ cultivar and early flowering peach (‘40A17’) produced an F1 where ∼50% of progeny showed signs of NBF, including canopy die-back, erratic branching patterns (known as “crazy-top”), and rough bark. In this study, whole-genome DNA methylation profiles were generated for three F1 progenies exhibiting NBF and three progenies considered NBF-free. Subsequent alignment to both the almond and peach reference genomes showed an increase in genome-wide methylation levels in NBF hybrids in CG and CHG contexts compared to no-NBF hybrids when aligned to the almond genome but no difference in methylation levels when aligned to the peach genome. Significantly differentially methylated regions (DMRs) were identified by comparing methylation levels across the genome between NBF- and no-NBF hybrids in each methylation context. In total, 115,635 DMRs were identified based on alignment to the almond reference genome, and 126,800 DMRs were identified based on alignment to the peach reference genome. Nearby genes were identified as associated with the 39 most significant DMRs occurring either in the almond or peach alignments alone or occurring in both the almond and peach alignments. These DMR-associated genes include several uncharacterized proteins and transposable elements. Quantitative PCR was also performed to analyze the gene expression patterns of these identified gene targets to determine patterns of differential expression associated with differential DNA methylation. These DMR-associated genes, particularly those showing corresponding patterns of differential gene expression, represent key targets for almond breeding for future cultivars and mitigating the effects of NBF-exhibition in currently affected cultivars.

Propagation of an Epigenetic Age-Related Disorder in Almond Is Governed by Vegetative Bud Ontogeny Rather Than Chimera-Type Cell Lineage

Almond (Prunus dulcis [Mill.] D.A. Webb) represents a model system for the study of epigenetic age-related disorders in perennial plants because the economically important noninfectious bud-failure disorder is well characterized and shown to be associated with the clonal-age of the propagation source. Epigenetic changes regulating disorders such as changes in methylation or telomere-length shortening would be expected to occur in shoot apical meristem initial cells since subsequent daughter cells including those in ensuing shoot axillary meristems show an irreversible advance in epigenetic aging. Because multiple initial cells are involved in meristem development and growth, such ‘mutations’ would be expected to occur in some initial cells but not others, resulting in mericlinal or sectorial chimeras during subsequent shoot development that, in turn, would differentially affect vegetative buds present in the leaf axils of the shoot. To test this developmental pattern, 2180 trees propagated from axillary buds of known position within asymptomatic noninfectious bud-failure budstick sources were evaluated for the disorder. Results demonstrate that relative bud position was not a determinant of successful trait propagation, but rather all axillary buds within individual shoots showed very similar degrees of noninfectious bud-failure. Control is thus more analogous to tissue-wide imprinting rather than being restricted to discrete cell lineages as would be predicted by standard meristem cell fate-mapping.

Relative Telomere Length and Telomerase Reverse Transcriptase (TERT) Expression Are Associated with Age in Almond (Prunus dulcis [Mill.] D.A.Webb).

While all organisms age, our understanding of how aging occurs varies among species. The aging process in perennial plants is not well-defined, yet can have implications on production and yield of valuable fruit and nut crops. Almond exhibits an age-related disorder known as non-infectious bud failure (BF) that affects vegetative bud development, indirectly affecting kernel yield. This species and disorder present an opportunity to address aging in a commercially relevant and vegetatively propagated perennial crop. The hypothesis tested in this study was that relative telomere length and/or telomerase reverse transcriptase (TERT) expression can serve as biomarkers of aging in almond. Relative telomere lengths and expression of TERT, a subunit of the enzyme telomerase, were measured via qPCR methods using bud and leaf samples collected from distinct age cohorts over a two-year period. Results from this work show a marginal but significant association between both relative telomere length and TERT expression, and age, suggesting that as almonds age, telomeres shorten and TERT expression decreases. This work provides information on potential biomarkers of perennial plant aging, contributing to our knowledge of this process. In addition, these results provide opportunities to address BF in almond breeding and nursery propagation.

Propagation from Basal Epicormic Meristems Remediates an Aging-Related Disorder in Almond Clones

The asexual propagation of clonal crops has allowed cultivation of superior selections for thousands of years. With time, some clones deteriorate from genetic and epigenetic changes. Non-infectious bud-failure (NBF) in cultivated almond (Prunus dulcis) is a commercially important age-related disorder that results in the failure of new vegetative buds to grow in the spring, with dieback of terminal shoots, witches-brooming of surviving buds, and deformed bark (roughbark). The incidence of NBF increases with clone age, including within individual long-lived trees as well as nursery propagation lineages. It is not associated with any infectious disease agents. Consequently, nursery practices emphasize the establishment of foundation-mother blocks utilizing propagation-wood selected from proven and well-monitored propagation-lineages. Commercial propagation utilizes axillary shoot buds through traditional budding or grafting. This study examines NBF development using basal epicormic buds from individual trees of advanced age as an alternative source of foundation stock. Results show the age-related progression of NBF is suppressed in these epicormic meristems, possibly owing to their unique origins and ontogeny. NBF development in commercial orchards propagated from foundation blocks established from these sources was similarly dramatically suppressed even over the 10- to 20-year expected commercial orchard-life. Foundation-stock stability can be further maintained through appropriate management of propagation source-trees, which requires accurate knowledge of meristem origin and development.

Epigenetic selection in almond breeding

Plant breeding targets the selection of desirable and heritable traits as a strategy for cultivar improvement. Sufficient heritability is required both for the efficient selection of desired genes within a diverse genetic and environmental background and, in seed-propagated crops, to ensure that all grower seed inherit the desired genetic composition. Clonal propagation avoids the requirement for seed progeny inheritance allowing for the direct selection of both genetic and non-genetic (epigenetic) interactions as long as the desired traits remain true-to-type following vegetative propagation. Utilizing Noninfectious-Bud-Failure (NBF) as a relatively well studied example of epigenetic inheritance in almond, methods for epigenetic selection both within and among genotypes have been successfully developed and employed, but remain tedious and time consuming. Molecular marker analysis utilizing methylation-sensitive amplified fragment length polymorphism (MS-AFLP) has documented extensive changes in epigenetic methylation patterns within both individual trees and individual clones. Both MS-AFLP and NBF patterns demonstrate change with both plant as well as clone age, which, while consistent with epigenetic control, confounded the detection of MS-AFLP markers diagnostic for NBF status. The preliminary study of genome-wide methylation profiles in almond identified exceptionally high level of epigenetic polymorphisms when compared to published results from seed-propagated crops. MS-AFLP status changed with both tree-age as well as clone-age, though patterns were detected which facilitate breeding selection. Results document both desirable as well as undesirable epigenetic variability existing within and among almond genotype/cultivars. Until more rapid diagnostics become available, long-term statistical assessment remains the only proven epigenetic selection method. A promising approach is to examine traits where the genetic control has already been identified but where inconsistent inheritance patterns supports the influence of epigenetic factors, as in the recent findings that methylation of the Sf locus in almond is associated with expression of self-compatibility through S-RNase loss of function.

Genome-wide DNA-(de)methylation is associated with Noninfectious Bud-failure exhibition in Almond (Prunus dulcis [Mill.] D.A.Webb)

Noninfectious bud-failure (BF) remains a major threat to almond production in California, particularly with the recent rapid expansion of acreage and as more intensive cultural practices and modern cultivars are adopted. BF has been shown to be inherited in both vegetative and sexual progeny, with exhibition related to the age and propagation history of scion clonal sources. These characteristics suggest an epigenetic influence, such as the loss of juvenility mediated by DNA-(de)methylation. Various degrees of BF have been reported among cultivars as well as within sources of clonal propagation of the same cultivar. Genome-wide methylation profiles for different clones within almond genotypes were developed to examine their association with BF levels and association with the chronological time from initial propagation. The degree of BF exhibition was found to be associated with DNA-(de)methylation and clonal age, which suggests that epigenetic changes associated with ageing may be involved in the differential exhibition of BF within and among almond clones. Research is needed to investigate the potential of DNA-(de)methylation status as a predictor for BF as well as for effective strategies to improve clonal selection against age related deterioration. This is the first report of an epigenetic-related disorder threatening a major tree crop.

Noninfectious Bud Failure in `Carmel' Almond: I. Pattern of Development in Vegetative Progeny Trees

The spatial and temporal pattern of noninfectious bud failure (BF) expression (BFexp) was studied during seven growing seasons in a population of `Carmel' almond trees originating from twelve commercial propagation sources. All progeny trees were grown in a single experimental site with high prevailing summer temperatures. BFexp increased continuously but irregularly in each nursery population as measured as the proportion of trees showing BF and as an average BFexp rating. Populations from the 12 nurseries represented increasing clonal generations from the original seedling tree and showed increasing levels of BF, as well as a decreasing shape value and increasing scale value derived by a failure statistics model. Models for development, distribution and hazard functions were defined for each of the 12 sources studied. Only sources from the original tree and source A demonstrated potential for commercial use. A significant correlation was found between average yearly increase in BFexp and the average daytime temperature for the previous June. The June period coincides with a specific stage in the seasonal growth cycle when vegetative buds mature.

REGIONAL ALMOND VARIETY TRIALS FOR CULTIVAR EVALUATION IN CALIFORNIA

Regional Almond Variety Trials (RAVTs) were planted in 1993 in Butte County at the California State University at Chico farm (Butte), in San Joaquin County at the San Joaquin Delta College farm near Manteca (San Joaquin) and in Kern County at a Paramount Farming Company orchard (Kern). Standard cultivars are planted 1:1 with new cultivars; ‘Nonpareil’ for the early-mid blooming cultivars and ‘Mission’ for the late blooming cultivars. Other standard cultivars planted include ‘Butte’, ‘Carmel’, ‘Fritz’ (not at Butte RAVT), ‘Monterey’, ‘Padre’, ‘Price’, ‘Sonora’. The new cultivars being tested in these trials are ’Aldrich’, ‘Chips’, ‘Donna’, ‘Dottie Won’ (San Joaquin RAVT only), ‘Kahl’, ‘Kapareil’, ‘Jenette’, ‘Jiml’, ‘Johlyn’, ‘Livingston’, ‘Morley’, ‘Plateau’, ‘Rosetta’, ‘Ruby’, ‘Sano’, ‘Savana’, ‘Wood Colony’ and ‘Yokut’. In addition six numbered selections from a University of California at Davis almond breeding program were included in these trials. These are 1-87, 1-102W, 2-19E, 2-43W, 13-1 and 25-75. Data collected from these trials include bloom time, hullsplit/harvest time, yield, and nut quality. Trees in these trials are also being observed and evaluated for growth characteristics, pest and disease susceptibility and noninfectious bud failure symptoms. The cultivars with the highest cumulative yields at the Butte RAVT are selection 13-1, ‘Plateau’, ‘Monterey’, ‘Carmel’ and ‘Nonpareil’. At the San Joaquin RAVT, the cultivars with the highest cumulative yields are ‘Carmel’, ‘Butte’, ‘Ruby’, ‘Plateau’ and ‘Fritz’. At the Kern RAVT, ‘Ruby’, ‘Plateau’, ‘Jenette’, ‘Padre’ and ‘Sano’ have the highest cumulative yields. Averaged across all three locations, the highest cumulative yields are from ‘Plateau’, ‘Ruby’, ‘Carmel’, ‘Monterey’, ‘Butte’, and ‘Jenette’. Of the high yielding cultivars, both ‘Plateau’ and ‘Sano’ have had a tendency towards a high percentage of double kernels in at least one of the trials each year. The variability in performance of the cultivars at the different RAVTs suggests that there may be some cultivars with qualities conducive to planting in particular regions.

489 Detection of Hidden Sectorial Chimeras in Almond Shoots through Distortions in Flower Symmetry

Somatic mutations in shoot growing points, while considered relatively common in many horticultural clones, cannot be detected unless the mutation results in a distinguishable change and comes to occupy sufficient shoot area to be observable. Noninfectious bud-failure (BF) in almond, a genetic mutation which results in failure during early development of vegetative but not flower buds, behaves as a chimera in its incidence within an affected tree and in vegetative progeny from bud-failure prone clonal sources (i.e., vegetative lineage). Early stages of BF development are thought to occur as very limited and so undetected sectorial or mericlinal chimeras. Detection of BF during these early stages would be very valuable for the selection of low-BF source clones for nursery increase. Flower symmetry, as measured by differences in the size of each of the five petals of an almond flower, was evaluated as an indicator of the relative fitness of the individual cell lineages from which different petals were derived. Several hundred flowers from individual clonal sources of the almond variety `Nonpareil', known to produce either very low, medium, or very high levels of BF in their vegetative progeny, were tested over 3 separate years. Significant reductions in flower symmetry were consistently observed for medium BF potential clonal sources relative to either low- or high-BF sources despite the lack of any observable BF symptoms in the medium-BF trees tested. Associated with asymmetric-flower-prone sources was a greater number of an easily distinguishable distorted petal morphology. Medium BF-potential sources consistently produced 2- to 3-fold greater numbers of this petal morphology relative to low BF-sources, although the occurrence of distorted petals in both low and medium BF sources limits its use as an efficient field selection tool. Research findings, however, are allowing a more detailed understanding of the developmental ontogeny of “bud-sport” mutations and may have application in the analysis of otherwise hidden chimeras resulting from either somatic mutations or genetic transformation/regeneration schemes.

063 Biological Basis for Noninfectious Bud-failure in Almond (Prunus dulcis)

Noninfectious bud-failure (BF) is a genetic disorder in almond, associated with nursery source selection. Previously (Kester, PASHS, 1968), the latent potential for BF (BFpot) was shown to be heritable but its phenotypic expression (BFexp) varied among individual seedlings of a populations as a function of age. Vegetative propagation perpetuates BFpot of individual propagules (Kester and Asay, JASHS, 1978b) but the subsequent age of BFexp within individual plants is a function of accumulated exposure to high summer temperature and growth (Kester and Asay, JASHS 1978a). A recent 7-year “somatic heritability” study of 12 commercial nursery sources (Kester et al., HortScience 1998abst) portrays the total range of variability of BFpot and BFexp within the entire `Carmel' almond clonal population and includes a pattern of BF increase in consecutive vegetative propagation cycles that mimics patterns produced by phase change (i.e., juvenile > mature) phenomena (Hartmann et al., 1997). Although phase change potential is heritable in seedling populations, phase change expression is not (Kester, HortScience 1983). Furthermore phase changes can be reversed under particular conditions during consecutive vegetative propagations (Hartmann et al., 1997). In contrast, evidence shows that BF produces permanent changes in genotype that are heritable and irreversable. High correlations exist between BFpot of individual source blocks, individual trees and individual budsticks and the age and severity of BFexp in progeny trees. The apparent continuous change in BFpot and BFexp within clones appears to be the pattern of expression of different populations of increasingly defective (?) somatic cells that result from consecutive sequences of change during annual cycles of growth and generations of vegetative propagation.

Final Results of Propagation Experiments to Show Distribution of Noninfectious Bud-failure in `Carmel' Almond

The distribution of the potential for noninfectious bud-failure (BFpot) was shown for the entire `Carmel' almond cultivar by propagating from single buds on single budsticks on individual trees of separate source blocks of commercial nurseries. Approximately 2700 progeny trees were grown at the same test site. The gradual increase in the expression of BF (Bfexp) in progeny trees from individual source trees shows a continuous range of latent BFpot among individual source trees directly correlated to five (or six) consecutive generations of vegetatively propagated source orchards. This pattern shows a continuous gradient in expression (vertical variation). A continuous gradient in expression also exists among progeny trees originating from the same source tree (horizontal variation). Individual source trees were identified whose progeny produced little or no BFexp when grown for 5 or more years in a high temperature test site. These studies confirm that noninfectious bud-failure (and probably other genetic disorders) represent persistent variants of a specific gene(s) whose genepot changes during vegetative growth sequences to bring about differential expression over time (“age”) and developmental patterns. These represent a class of clonal variants which are “age-related” and include “phase changes”, “juvenility–maturity” phenomena, and can affect morphology, thorniness, ease of rooting, etc. BF is related to a gene that affects environmental adaptation of the almond. Control is through the selection, maintenance and management of foundation clones.

The Concept of “Foundation Clones” in Source Selection for Vegetative Propagation of Almond

The term “clone” is a key biological term that designates a number of horticultural situations. In breeding, many cultivars are designated as clones, originating from consecutive vegetative propagation from individuals within a seedling population, from individual plants of a clone exhibiting “bud mutations,” and, more recently, from genetic engineering and biotechnology. Extensive vegetative propagation of a limited numbers of clones in modern horticultural systems has been accompanied by systemic incorporation by serious pathogens (viruses, viroids, phytoplasmas, etc.), and in some cases by horticultural deterioration (e.g., noninfectious bud-failure in almonds). Control of these problems in clonal propagation is achieved by 1) propagation source selection 2) maintenance of the source in a registered foundation block under protected conditions and 3)multipli-cation in controlled “mother blocks” or “increase blocks” from which commercial material is distributed after a minimum of consecutive generations of vegetative propagation. This system is the basis for Registration and Certification programs and “clean stock” in general. In many crops the selected propagation source is a single plant, its progeny constitutes a “clone,” and the new entity is given a unique name or number. To distinguish this “new” clone from the “original” clone, the designation of FOUNDATION CLONE is suggested. Biological and horticultural significance is illustrated in almond (Prunus dulcis).

Potential for Noninfectious Bud-failure in `Carmel' Almond Orchards in California

The potential for noninfectious bud-failure in propagation source material for `Carmel' almond in California has been determined in progeny tests from commercial nursery sources. Percentage BF increased with time (temporal), but decreased in severity (spatial). Analysis of variability in nursery sources showed that the key to successful selection for low BF potential is the individual tree, although variability exists among nurseries, budsticks (within trees), and individual buds (within budsticks). One-half of the individual trees of the nursery population tested have produced BF progeny so far within the test period. Future BF from the remainder was project by a BF model to be beyond the critical economic threshold. Two low BF-potential single tree sources were identified for commercial usage and progeny tests have started on an additional 19.

VERIFICATION OF AN EPIDEMIOLOGY MODEL OF NONINFECTIOUS BUD-FAILURE IN `CARMEL' ALMOND

A model for the epidemiology of noninfectious bud-failure (Fenton, et al., 1988) predicts that BF-potcntial is universally present within specific almond cultivars with variation existing in the rate and pattern of development of BF phenotypes. Orchard surveys of Carmel in 1990 and 1991 involving four nursery sources showed a trend of 2 per cent of affected trees after one year in the orchard, increasing to 4 per cent in the second, with prospects for gradual increase with time. All four sources produced some BF trees with significant differences among sources. A study has been started to identify the source and pattern of BF-potential within the entire Carmel cultivar. It has two parts. A pedigree analysis of propagation sources from eleven commercial nurseries traces their genealogy from the original seedling plant first discovered in 1947. A propagation test of approximately 3000 individual trees representative of the propagation sources of all eleven commercial nurseries has been established. The origin of each progeny tree has been maintained in respect to source, tree, budstick and individual bud location on the stick. Expression of bud-failure symptoms in individual trees will identify the source and pattern of BF-potential within the cultivar.

Yield comparison of Australian selections of almond cv. Nonpareil

Eight Nonpareil almond clones on Nemaguard rootstock were yield-tested in a field trial at Irymple, Victoria, for 4 cropping seasons (1986-89). Although few significant differences in yield were recorded in any year, the results showed that the Nonpareil clones differed in total yield and in total yield per unit of butt cross-sectional area by as much as 50%. For example, clone L54/3 produced a total dry kernel yield per tree per unit of butt crosssectional area of 51.2 kglcm2, while the high yielding clone G11/16 produced a yield of 77.6 kg/cm2. The 3 top-yielding clones (GI 1/16, P7/29 and K5/7) produced total dry kernel yields (1986-89) per tree per unit of butt cross-sectional area of 77.6, 73.6 and 71.7 g/cm2. However, clone K5/7 proved to be subject to non-infectious bud failure, and it is concluded that clones G11/16 and P7/29 could be useful, high-yielding clones for the Australian almond industry.

Search for a Viroid Etiology for Noninfectious Bud Failure in Almond

Abstract Viroids could not be detected in nucleic acid samples of almond [Prunus dulcis (Mill.) D.A. Webb] with noninfectious bud failure by one- and two-dimensional electrophoresis. Almond nucleic acids showed no detectable hybridization with a 32P-labeled potato spindle tuber viroid (PSTV) cDNA probe. PSTV controls were included.

The temperature response of cultured almond cells derived from tree sources with and without non-infectious bud failure. I. Specific growth rate

The specific growth rate of ‘Nonpareil’ almond cell cultures growing in exponential phase was measured at temperatures in the range of 20–30°C. Specific growth rates differed with the non-infectious bud failure (BF) potential of the source plant, the cell line, the age of cell lines, and the temperature. The optimum temperature for growth was found to be in the range of 28–32°C. Specific growth rate decreased more sharply at temperatures above the optimum for the cell lines derived from BF sources.