Frogsleap Farm

Frogsleap Farm

Monday, April 24, 2017

Use of Molecular Markers in Tomato Breeding

Foolad and Panthee (2012) give an excellent review of Marker Assisted Selection (MAS) in tomato breeding, providing more detail than intended here.  Our goal is to explain the basics and give examples of how this tool can be used by tomato breeders, and how we are employing the technology in our breeding program.

There are various types of molecular markers that can be used in breeding (e.g. SNP, AFLP, SCAR, CAPS and SSR), but these all represent a short DNA sequence with a known physical location in the genome – essentially “mile markers” on the roadmap that defines structural characteristics of the plant DNA sequence.  For example there are tens of thousands of SNP markers now placed on the physical map of the tomato genome.  When one of these markers is adjacent to (or within) a gene of interest, such as a gene associated with resistance to a particular pathogen, the marker and the gene co-segregate – and selection for the marker is an effective surrogate for selection for the trait. 

Distribution of SNP markers on tomato genetic map (Viquez-Zamora et al., 2013)

PCR-based genotyping can be used to quickly confirm presence or absence of a particular molecular marker, and some marker types (including SNPs) can determine whether the plant contains one (heterozygous) or two (homozygous) copies of the marker.  These assays are conducted on DNA extracted from a very small amount of plant tissue, and thus can be performed on very young seedlings.

Leaf samples in 96 well plate for PCR-based genotyping (photo: U of Georgia)

Some of the advantages are obvious:

1)   The presence the molecular marker can be confirmed at the seedling stage, and not reliant on a trait phenotype that is expressed several weeks later (e.g. fruit color), or on a phenotype that is dependent on particular environmental conditions (e.g. disease resistance).   Early selection allows early culling of undesirable plants based on genotype rather than phenotype.  Early culling means that only those plants pre-selected for the desired trait or combination of traits go to field breeding nurseries or crossing programs.
2)   Trait stacking is the breeding process for combining multiple desirable traits into a single breeding line.  The first step always involves using crossing to bring the multiple traits into a single breeding population.  The second step, greatly aided by MAS, is to identify low frequency plants in the breeding population that contain all the traits of interest.  For example: the cross AABBCC x aabbcc, where genes A and B control resistance to two independent tomato pathogens (e.g. ToMV and LB) and resistance is dominant, and c is a desirable recessive allele for a trait for fruit color (e.g. ogc/crimson).  The F1 progeny will all be AaBbCc and in the F2 only one plant in 64 will have the desired stable genotype AABBcc.  Now think about stacking 7-8 genes  (which we are) - MAS allows the efficient testing of hundreds of F2 progeny to find the “needles in the haystack” combing the desired traits.  When stacking more than 3 traits it will likely be necessary to do this in a stepwise fashion.

How is MAS being used today?
Molecular markers have now been identified for many of the multiple genes associated with resistance to key tomato diseases and nematodes, and in some cases to the multiple races of the diseases now prevalent (e.g, all three races of Fusarium wilt).  Commercial breeders have been successful at stacking resistance to most of these key pathogens in newer hybrids – with MAS being a critical tool in such stacking.

Molecular markers have also been identified for a handful of other major genes controlling important traits – but it is a short list:  tomato fruit color - red vs yellow and crimson vs red; fruit size - faciated; plant type - determinant vs indeterminant; and presence of the rin allele associated with delayed ripening.  Some of the major genes controlling plant/fruit phenotype are shown below (photo courtesy of University of Newfoundland) - linked molecular markers are available for a few of these.

Map of major genes controlling plant/fruit phenotype

All of the examples cited above involve major genes providing control of simply inherited traits.  However we know that many important traits are quantitative traits, controlled by multiple genes – typically each with a small, but cumulative effect.  Quantitative trait loci (QTL) are molecular markers associated with genes/alleles contributing to a quantitative trait – such a fruit yield, fruit size and fruit flavor.  MAS allows the effective stacking of QTLs that in concert have a major impact on a “hard to breed for” quantitative traits.

Harry Klee and his colleagues at the University of Florida have been working to unlock the mystery of flavor in tomatoes.  What are the multiple components of flavor, how do they interact, and what is the genetic control of these factors?  I am confident this will eventually lead to QTLs associated with key flavor components – facilitating breeding for better tasting tomatoes.

University breeding programs and the larger commercial tomato breeding companies all have access to the tools required for MAS.  Thankfully there are also a few commercial companies that conduct such activities on a fee for service basis, which allows the smaller players (FLF included) access to these tools.  Such access is a significant incremental expense per se, but allows cost savings, and an accelerated timeline in the long run.

Our breeding program started with crosses between heirloom types with a primary goal to improve flavor and plant health.  After a couple years we started crossing these to a handful of commercial types, including a couple of the NCSU hybrids, to introduce improved disease tolerance and improved fruit quality.

Disease resistant X Great Flavor

 We are now using molecular markers to identify progeny from these crosses which combine resistance to multiple pathogens – and will follow that genotypic selection with phenotypic selection for flavor and fruit quality in our various breeding nurseries.  The use of molecular markers and our recent access to facilities allowing 3 breeding generations/yr should allow us to soon commercialize new F1 hybrids with state-of-the-art disease resistance, best-in-class heirloom flavor, and in a rainbow’s array of colors and stripes.

MAS-enabled multiple disease resistant F4 - a F1 parent "in training"

Sunday, November 22, 2015

Breeding Strategies for Improving Shelf Life in Tomatoes

Tomato is one of many plants that have evolved an “edible fruit” strategy for seed dispersal.  Mature seed is encased in a fruit designed to be attractive for consumption by fruit eating animals.  Seed dispersal occurs when the consumed seed passes safely through the digestive tract and is deposited with feces on the soil some distance from the mother plant.  In tomato the fruit ripening process involves several steps designed to enhance attractiveness for consumption:  an increase in fruit sugars, acids and flavor-enhancing aromatic compounds that greatly improve tastiness of the fruit; fruit softening to a more edible texture; and obvious fruit pigmentation designed to signal to passing animals that the fruit is fully ripe and ready to eat.  These features were preserved during the domestication of tomato and the more recent development of tomato as one of the world’s most important fruit/vegetable crops.

One of the modern dilemmas in tomato production and breeding relates to managing post-harvest losses associated with the modern agricultural practice of concentrated fruit production in one area and fruit consumption in another place (and time).  Ripe fruit is easy to damage in transit and deteriorates relatively quickly.  Picking mature green (MG) fruit for shipment and gassing with ethylene at a distant delivery point to “ripen” the fruit solved the problem of damage in shipping, but comes with an unfortunate sacrifice in flavor.  As an alternative to this practice plant biologists and tomato breeders have looked at various genetic variants (mutations) in genes controlling the ripening process, and examined how these novel alleles might be deployed in the development of varieties with great flavor and enhanced shelf life.  In this post I’ve tried to summarize the current understanding of this field and share some of our related breeding efforts.

Tomato Fruit Development (from Alba et al., 2005)

The Ripening Process
Tomatoes are a climacteric fruit, which means that the plant hormone ethylene is required for fruit ripening.  Ethylene is rapidly produced in tomato fruit at the breaker (BK) stage and drives a series of reactions that together define the fruit ripening process.  During normal ripening there are simultaneous and independent processes that lead to 1) accumulation of sugars, organic acids and volatile organic compounds influencing flavor, 2) conversion of chloroplasts to chromoplasts and the synthesis and accumulation of carotenoid pigments and 3) softening of the fruit.  In a perfect modern tomato, ripening steps 1&2 proceed normally and step 3 proceeds at slow rate – allowing the tomato fruit to keep peak flavor, color and texture for an extended period of time.

ESL, or extended shelf life, is a term describing a collection of traits that together extend the potential time between picking of fully ripe or nearly fully ripe fruit, and the deterioration of fruit quality.  Fruit quality deterioration is usually associated with fruit softness/undesirable texture and fruit rotting.  Taste panels have identified fruit texture as an important determinant in consumer preference, and soft or mealy fruit is a major “turn-off”.  Deterioration in fruit firmness/texture is generally associated with a ripening related spike in polygalacturonase (PG) and other enzymes that degrade fruit cell wall polysaccharides. Thus, a decline in fruit firmness typically coincides with dissolution of the middle lamella and hemicellulosic/pectic cell wall polysaccharides, thereby undermining the polysaccharide network that hold cells together in the fruit pericarp.  FlavrSavr tomato, the commercially unsuccessful GE trait introduced by Calgene in 1985, was designed to specifically suppress PG activity in ripening tomatoes.  Recent research has also implicated cuticle composition and architecture as traits influencing ripening-induced fruit softening (Saladie et al. 2007 and Kosma et al. 2010).  The cuticle has long been implicated as a contributor to fruit strength, and cuticle structure changes during the ripening process.  Kosma et al, show that during the ripening process ESL mutants generally have cuticles with mechanical properties significantly different than the wild type – likely contributing to ESL per se.

It should be noted that independent of the several novel mutant alleles described below, there are significant genetic differences in firmness in tomatoes.  Unfortunately there are a couple of studies that report fruit firmness at harvest is not well correlated with the maintenance of fruit firmness postharvest.  We have found that pericarp thickness, relative to size of the locules, is a heritable trait that significantly impacts firmness per se, and appears in many cases to be associated with improved shelf life (see photos below).  This combination of traits is common in many newer commercial hybrids.

Firm when ripe phenotype

There are several mutations in key structural or regulatory tomato genes that affect the ripening process.  These genes generally either inhibit ethylene synthesis and/or modify ethylene’s downstream effects on specific biochemical processes related to fruit ripening.  To better understand climacteric fruit ripening per se, and to examine the potential utilization of these mutant alleles for delayed ripening/extended shelf life – tomato scientists have characterized several mutant alleles associated with a delayed ripening phenotype.  Several key ripening mutants are described in detail below.

Key genetic mutations affecting tomato fruit ripening
rin = ripening inhibitor.  The RIN gene is a transcription factor that acts as a master regulatory gene controlling numerous genes and pathways associated with tomato fruit ripening.  The rin loss of function mutant is a recessive allele that both represses genes associated with ethylene synthesis and modifies downstream processes associated with the normal ripening process.  Specifically rin modifies expression of other transcription factors associated with fruit ripening (e.g. NOR); prevents normal fruit pigmentation by suppressing synthesis of Phytoene synthase (PSY), the primary enzyme regulating flux into the carotenoid pathway (see Genetic Control of Fruit Color in Tomatoes); suppresses key steps in the accumulation of sugars, organic acids and aromatic compounds associated the improved flavor in ripe tomato fruit; suppress enzymes (e.g. polygalacturonase = “PG”) associated with breakdown of cell wall polysaccharides that lead to ripening-related fruit softening; and modifies cutin and fruit wax content and composition.   The rin/rin homozygote plant produces fruit that never fully ripen and have much firmer fruit with a significantly longer shelf life (see photo below).  The lack of normal color and flavor significantly limits commercial potential of rin/rin plants.  In the heterozygous condition rin/+ plants produce fruit with near normal fruit color and flavor, and shelf life that is intermediate between rin/rin and +/+ (wild type) plants. F1 hybrids with the rin/+ genotype and extended shelf life have been widely commercialized and are a key driver in the recent availability of “vine ripened” tomatoes in grocery stores.  The extended shelf life allows picking at or near the full ripe stage when flavor is near peak, and remaining firm for an extended period of time for shipping to distant locations.

We have been developing and testing new rin/rin inbreds and rin/+ hybrids for the last few years. 
Striped rin/rin cherry
Although rin/rin lines generally have very low fruit sugars, there are differences in sugar levels between rin/rin lines.  The sweetest rin/rin lines generally produce the sweetest rin/+ hybrids, though this is also heavily influenced by the non-rin parent in the hybrid.  Lycopene levels in rin/+ hybrids is a little lower than wild type (orange/red vs dark red), but normal red color can be restored in ogc/ogc crimson types (e.g. Mountain Magic).  Enhanced shelf life in rin/+ hybrids appears to be influenced by rin per se, but also on the genetic background of the rin and wild type parents, specifically those genes influencing fruit firmness.  Ripening is a little slower with rin/+ hybrids, adding perhaps 5-7 days.  We are making great progress on rin/+ hybrids and it appears possible to combine a significant improvement in shelf-life with exceptional flavor in fruit in a wide range of colors, shapes and sizes. 

Fruit at BK +7 stage (7 days after breaker stage in the WT)

                      Wild Type                     rin/rin                        nor/nor
Photo by Martel, 2010

nor = non-ripening.  The NOR gene is an unrelated transcription factor that also serves as a master regulator of fruit ripening in tomato.  The recessive loss of function mutant allele nor has been widely studied.  The nor/nor homozygote has a very similar phenotype to rin/rin, and nor/+ hybrids also have much restored color and flavor with extended shelf-life – though reports in the literature suggest less color and flavor and longer shelf life in nor/+ relative to rin/+.  The specific mechanisms for modification of ripening in nor mutants is less understood than with rin – but like RIN, NOR helps regulate multiple genes and pathways important in tomato fruit ripening.  Commercial nor/+ hybrids have been commercially successful, though probably less so than rin/+.  Note that the next few mutants described here, alc and dfd, are thought to be allelic to nor (i.e. independent NOR mutants) with subtle but significant differences in ESL phenotypes.

alc = alcobaca.  The Spanish tomato landraces Alcobaca, Penjar and Tom├átiga de Ramellet are generally “long keeping” types with much delayed fruit deterioration.  These landraces have been selected for hundreds of years for local adaptation to a dry climate and for fruit that will have acceptable quality for months after harvest.  The photo below shows a typical fall/winter storage strategy employed in the region – fruit are hung in small bunches for medium term storage.  Note the term tomatiga de penjar means tomato for hanging.  There is a single recessive allele “alc” associated with the slow ripening phenotype.  The alc allele is believed to be another mutation at the NOR locus.  Fruit from alc/alc plants have significantly lower levels of endogenous ethylene, suppressed polygalacturonase activity and firmer fruit.  Fruit harvested at the onset of ripening mature to an orange color, and those left on the plant until full ripening have normal red color.  The landraces listed above are all alc/alc and can remain firm for several months, though there is wide variation for this LSL trait within local populations – suggesting alc + other factors are at play.  The ESL trait associated with alc also appears to be subject to the level of water stress during fruit production – with generally enhanced ESL under more arid production conditions.  Hybrids that are heterozygous for alc (alc/+) have shelf life intermediate between +/+ and alc/alc, but have more normal fruit color and flavor than either rin/+ or nor/+, and thus seems to be another interesting candidate gene/all ele for the extended shelf life/excellent flavor combination.

Alcobaca type tomatoes hung for winter storage

Effect of alc on fruit deterioration

dfd = delayed fruit deterioration.  The dfd trait was first found in certain ecotypes growing in the southern Mediterranean.  The literature suggests that dfd is a partially dominant mutant allele of NOR, and may indeed by identical to or a slight variation to alc.  DFD controls cuticle composition and leads to decreased cell water loss, increasing cell turgor (firmness) per se, and decreasing fruit water loss generally during ripening.  Normally as tomato fruit ripen the cuticle weakens and grows less resistant to penetration.  Fruit of dfd plants require significantly more force for cuticle penetration than those from wild type varieties, and do not exhibit a normal progressive weakening of the cuticle during ripening.  Fruit from dfd plants exhibit the normal ripening-induced fruit cell wall breakdown and cell separation typical of wild type, but show substantial swelling of pericarp cells during the ripening that is atypical, with a ~4x increase in cell size vs wild type in ripe fruit, likely related to increased cell turgor.  There is also less fruit water loss in dfd vs wild type ripening fruit – another contributing factor to improved fruit firmness.  Increased cell turgor, decreased fruit moisture loss and increased cuticle strength all appear to be related to changes in cuticle wax content and composition in dfd vs wild type.
Unlike rin, and nor, dfd’s affect on fruit firmness/LSL was independent of normal fruit coloration and ripening-related accumulation of sugars and organic acids.  Futhermore dfd/dfd plants maintained firmer fruit without impacting expression of genes, such a PG, involved in ripening induced cell wall degradation (unlike alc).  The dfd mutant appears to represent a very novel approach for ESL that may be used in combination with other ESL traits to enhance shelf life in tomato hybrids or O.P. varieties.

Changes in Fruit Coloration after Breaker Stage

Davis EFS F2 segregate 
EFS – extended field storage.  Several new processing type tomato hybrids contain the extended field storage (EFS) trait, which allows for a longer window for field harvest, creating more flexibility for tomato processers.  The alc allele (or perhaps a related NOR mutant) may to be at least partially responsible for this modified ripening phenotype.  While driving near Davis, California in early September 2014, I stopped to pick up a couple of tomatoes that had fallen off a truck on the way to processing.  They had bright crimson flesh and a rich tomato flavor.  In a F2 growout in 2015 we found one F2 plant that appeared never to fully ripen on the vine, but had a bright pink center (see photo).  This combination of a lack of obvious pigmentation on the fruit surface with bright lycopene pigmentation of the fruit pericarp seems atypical of all the ripening mutants described above, and remains a mystery.  We presume this plant to be homozygous for one or more recessive ripening mutants and made several F1 crosses to elite FLF breeding lines.  F2 progeny from winter growouts will be evaluated in 2016.  This was one of the oddest discoveries in our 2015 nurseries and I expect we will learn quite a bit more next year.  In my literature review for this paper I found a one sentence reference to a long keeping variety that appeared to ripen from the “inside out” – perhaps a related phenomenon?

Fruit Shelf Life of Nine LSL Tomato Hybrids (Yogendra et al. 2013)

Nr = never ripe and Gr=green ripe.  These are dominant, gain of function mutations at independent loci, that each results in reduced ethylene responsiveness in tomato fruit tissue.   The ethylene insensitivity in both Gr and Nr have a negative impact on seed germination and seedling vigor and completely prevent normal fruit ripening.  Negative plant and fruit phenotypes prevent any commercial use of these mutant alleles.

Although the mutant alleles rin, nor and alc generate a somewhat similar ESL phenotype in plants heterozygous for these alleles, rin and nor are independent loci and all three have different modes of action. With all three alleles, extended shelf life is associated with later maturity, and with rin and nor also associated with decreased pigmentation (see photo above).  The mutant alleles of these three genes have a similar effect on extending shelf life, and the maintenance of firmness is due both to the mutant alleles per se, and the background genotype of both the male and female parents.  We have found that a rin/+ genotype in a firm fruited background can extend shelf life for over two weeks.  In such a case a fruit picked fully ripe can stay crisp and firm on the countertop (or in transit to local or distant markets) for at least 14-21 days.  Since several of the key aromatic compounds impacting flavor are directly derived from lycopene and other carotenoid pigments, in theory one might expect that the lower carotenoid pigment content of rin/+ hybrids might lead to lower flavor.  However by selecting ruthlessly for flavor in parent lines, we have been able to identify rin/rin parents that contribute high flavor to rin/+ hybrids. 

It is currently unclear how closely related are the NOR mutants alc, dfd  - and possibly EFS.  EFS is now widely deployed in commercial processing hybrids grown in California, though the ESL phenotype and mode of action appear to be treated as trade secrets.  The dfd mutant is also somewhat of a mystery, perhaps due to a Cornell patent filing on a specific dfd sequence – in the patent they do describe this as a NOR mutant derived from a Mediterranean ecotype.  To complicate matters more a Davis, CA company Arcadia has patented an induced mutation in NOR (reference), which they claim to be an improvement on the naturally occurring nor loss of function mutant.  It is too early to know how similar the Arcadia mutant might be to alc, dfd or EFS.

The primary use of extended shelf life (ESL) tomato hybrids will likely be for medium/large size grower (field or protected culture) producing for distant markets.  Picking an ESL hybrid at or just before full ripening (in the marketplace = vine ripened) then packing and shipping, can be a consumer and taste-friendly alternative to the traditional “green and gassed” model.  We think ESL types will also be well suited to smaller producers selling in more local markets.  These types could be picked less frequently, and once picked, be much less prone to post harvest losses.  It appears there may be several different gene/allele options for ESL, with varying efficacy, ease of use, and freedom to operate.  We think ESL will be an increasing important trait for fresh market tomatoes, with perhaps evolving breeding strategies for optimization of the trait.  We will build on our early success with rin, and continue to follow and explore the other options described here.  Our multi-year effort in selecting for fruit firmness and flavor per se is paying off – deployment of rin or one of the NOR mutants will likely require a firm fruit background for optimization of ESL, and a high flavor background will likely be needed to counter the delayed ripening effect of rin/+, nor/+,  or alc hybrids.