Specialty Crop Germplasm and Public Breeding Efforts in the United States

Abstract

The United States Department of Agriculture/Agriculture Research Service/National Germplasm System (USDA/ARS/NGS) plant germplasm collections contain more than 600,000 different accessions of 16,289 species, including virtually all the ethnic and specialty crops of interest to United States (U.S.) agriculture. These and additional collections of plant seeds and asexual propagules are maintained at various laboratories and facilities geographically dispersed in the U.S. including NGS and many public land grant university institutions. The majority of these species fall under the definition of “specialty” crops since their utility is either narrow in scope or has not been fully developed. This paper summarizes the status of “specialty” and ethnic crop species germplasm in the U.S. including where the collections are maintained and how they are being used.

1. Introduction

The USDA defines specialty crops as “fruits and vegetables, tree nuts, dried fruits, horticulture, and nursery crops (including floriculture)” (USDA Specialty Crop Definition www.ams.usda.gov/sites/default/files/media/USDASpecialtyCropDefinition.pdf) (accessed on 10 November 2021). For the purposes of this paper, the scope of crops considered to be “specialty” will be further restricted to horticultural food crops that are produced and consumed on a smaller scale than in the mainstream. For example, potato, sweet corn, peach, apple, tomato, etc., are mainstream crops while vegetable greens, asparagus, small fruits, and spices are “specialty”. Demand for diversity and culinary quality in the food supply has surged over the past 20 years, driving a progressively larger acreage of specialty crop farming, processing, and distribution [1]. This demand has placed new pressures on the agricultural systems to enable production, including cultural practices, plant breeding, and postharvest handling and packaging. This paper will focus on public breeding efforts in the U.S. and sources of germplasm to meet the growing demands of these breeding programs. Where it makes business sense, the private sector is also developing new cultivars of specialty horticultural food crops, but access to information on these efforts is tenuous.

Demand for new cultivars begins with consumers who shift food purchases in a new direction. The market responds accordingly but, in the case of specialty horticultural food crops, markets are often insufficient to cover research and development costs and meet the profit needs of private businesses. The overarching benefits of specialty crops in the diets of consumers drive the establishment of research and development in the public sector where these costs are subsidized with tax dollars. In many cases these public sector efforts culminate in expanded markets that render it more attractive for the private sector. A good example is asparagus where most research and plant breeding were confined to the public sector until the late 20th century when seed companies progressively assumed more importance due to larger markets and the profitability of all-male hybrids [2].

Plant breeding programs are similar to manufacturing in that they depend on raw materials that are crafted into a value-added product through a defined process. The raw material to the plant breeder is the pool of genes that must be identified, isolated, and melded into the genome of a new genetic entity that expresses a unique and desirable phenotype. New molecular technologies have made it progressively easier and less costly to work directly with genes extracted from genomes but in 2021 it is still impossible to assemble a genome from scratch from a set of isolated genes. Consequently, plant breeders still employ strategies that were developed during the early 20th century based on controlled mating and selection to cut and spice genes from different plants into a new cultivar. Molecular technologies have provided new tools to make this process faster and more precise, for example marker-assisted selection [3]. Perhaps the future will come with more powerful lab methods to develop new cultivars of plants but we will rely on permutations of these archaic methods for some time to come.

The first plant breeders were the farmers at the dawn of agriculture as human societies became tethered to the land and domesticated plants and animals for food and other human needs. Primitive farmers selected plant from wild habitats to cultivate and ensuing generations continued this process of selecting the most desirable plants as sources of seeds and propagules for future years. Over the course of 20,000 years of mass selection to the start of the 20th century our populations of domesticated and refined crops were developed and were maintained by local farmers [3].

The 20th century brought massive changes to the plant breeding process. The principles of inheritance were described and extended and, ultimately, applied to plant breeding. This resulted in a monumental acceleration of the rate of progress in plant breeding and the development of new population concepts, for example hybrid cultivars. Seed companies sprang up and capitalized on these advancements since local farmers lacked the ability to seize on technology. Simultaneously in the U.S. the system of agricultural research and extension in the new land grant university system was revolutionizing agriculture with applications wrought by the industrial revolution [4]. Plant breeding was adopted as a new branch of this system. Between 2000 and 2020, specialty vegetable crop yields grew by an average of 37.2% in the United States (FAOSTAT 2021). The highest yield increases occurred among cabbages and other brassicas (114.4%), watermelons (75.5%), other melons including cantaloupes (54.6%), chilies and peppers, green (51.3%), and sweet potatoes (50.7%) (

Table 1. Yield increases of selected specialty crops in the United States between 2000 and 2020. (Data extracted from FAOSTAT https://www.fao.org/faostat/en/#data/QCL) (accessed on 3 December 2021) 50% of total yield increase may be attributed to crop breeding [5,6]).

Specialty Crop	2000	2020	Yield Increase (%)
	Yield (kg/ha)
Beans, dry	1840	2204	19.8
Cabbages and other brassicas	23,650	50,717	114.4
Carrots and turnips	38,562	56,094	45.5
Cauliflowers and broccoli	16,404	21,834	33.1
Chick peas	1421	1822	28.2
Chillies and peppers, green	24,039	36,365	51.3
Eggplants (aubergines)	33,459	36,888	10.2
Lentils	1587	1616	1.8
Maize, green	15,331	20,452	33.4
Melons, other (inc.cantaloupes)	23,076	35,686	54.6
Okra	7641	7835	2.5
Onions, dry	48,375	71,100	47
String beans	8743	10,370	18.6
Sugar beet	58,565	65,972	12.6
Sunflower seed	1501	2007	33.7
Sweet potatoes	16,292	24,553	50.7
Watermelons	25,365	44,504	75.5
		Mean Yield increase (%)	37.2

). It is estimated that 50% of these yield increases were due to plant breeding [5,6].

Exploration of the world during the 15th to the 19th centuries revealed a trove of new life forms that could be distributed throughout the world and produced locally. Exploration also invoked the notion of plant and animal collections. By the mid-20th century, the U.S. Department of Agriculture assumed the responsibility of establishing and maintaining collections of the world’s plants and animals and making them available to breeders and other researchers. This effort has grown into a large organization under the Agricultural Research Service (ARS) called the “National Germplasm System” (NGS). The collections are documented by a database, the “Germplasm Resources Information Network” (GRIN) that is accessible on-line to breeders and researchers to scrutinize and make specific requests for seeds and propagules. USDA/ARS/NGS performs evaluations and other studies of this germplasm within ARS, but most of the utilization of accessions occurs within the land grant university system and by private organizations such as seed and chemical companies [3].

A clear trend towards the growth of plant breeding activities in the private sector and decline in the public sector has been documented by several studies. Shelton and Tracy [7] concluded the following: public cultivar development is in a state of decline, with insufficient numbers of younger breeders working in the public sector today to maintain the current level of cultivar development as the most senior breeders retire. Funding public breeding programs continues to be a challenge, as is access to improved germplasm due to overly restrictive licensing agreements. Potential opportunities include re-distribution of royalty funds to bolster revenue streams and simplifying the germplasm exchange process to increase the likelihood of successful cultivar releases. Coe et al. [8] found that public plant breeding programs had experienced significantly declining hours spent on breeding activities by program leaders and technical support staff. Funds invested into public plant breeding activities also declined substantially over recent years.

This paper summarizes the sources of specialty horticultural food crop germplasm available from USDA/ARS/NGS, where and what crops are being addressed by the land grant university system in the U.S., and how germplasm has contributed to the progress and effectiveness of plant breeding. The impact of germplasm extends far beyond the realm of plant breeding. Genetic variability also provides a powerful tool to elucidate principles of biology.

2. Specialty Crops Breeding in the U.S. Public Sector

As the U.S. land grant university system grew after they were first established in 1862. The Morrill Acts of 1862 (12 Stat. 503) and 1890 (26 Stat. 417), and the Equity in Educational Land-Grant Status Act of 1994 (P.L. 103-382 §531-535), established the three institutional categories of the land-grant system, now known as the 1862, 1890, and 1994 Institutions [9]. During the early 20th century as the science of genetics was born and quickly took root in academia, the land grant system recognized applications in the more rapid and precise development of crop cultivars. The first model system for agricultural genetics was maize, but genetics was quickly integrated into cultivar development for other important food and fiber crops such as wheat, barley, oats, rice, cotton, and soybean. Horticultural crop species, and particularly those of “minor” importance, were mostly ignored until the latter half of the 20th century. Ubiquitous horticultural crops such as tomato, sweet bell pepper, onion, potato, lettuce, cabbage, carrot, and cucumber were progressively addressed by public plant breeders and researchers while many less prominent species still struggle in 2021 to gain traction. The demand for culinary quality and diversity has prompted more attention to the so-called minor or “specialty” horticultural food crops such as spices, Cuphea, chili peppers, leafy greens, and other small-acreage alternatives.

The land grant university system has responded to this demand by consumers. While many land grant institutions sponsored plant breeding programs in major food crops starting in the early 20th century, they were reticent to allocate tax dollars to crops considered by many to be marginal. The first position for the author after finishing graduate studies was as a breeder of celery in California. The University of California Davis, Department of Vegetable Crops (now Plant Science) was charged with establishing this position in a relatively minor food crop after the industry rallied behind a new disease (Fusarium yellows) challenge that necessitated a plant breeding solution. The celery industry coalesced under the umbrella of an Advisory Board that raised funds through industry assessment to pay for this new position. This position still exists at UC Davis in 2021 and is still funded by the California celery industry. Over the decades many other challenges facing celery growers were also addressed by this source of financial support. This is an example of how research and development into specialty food crops is begun and perpetuated in the public sector. The establishment of new programs and faculty positions to oversee them rarely comes without prodding from an external force.

Progressively, the forces of technical need filter into public research institutions from the constituencies they are charged with serving. Ideally the land grant institution effectively reflects the agricultural economy of the state in which it resides. If the state is mostly a monolith of wheat such as Kansas, the land grant institution (Kansas State University) tends to focus most of the collective efforts on solving problems faced by wheat growers. Many states have evolved into a circumstance where many diverse agricultural industries compete for the attention of the resident land grant institution, such as California, Texas, and Florida. Other states with smaller agricultural economies have also experienced increasing agricultural diversity that is driven by local market opportunities and demands and natural resources (e.g., soils and climate).

An analysis of the current allocations of positions to plant breeding assignments in specialty horticultural food crops, as defined above, is summarized in

Table 2. A partial list of public breeding programs focusing on specialty crops within the U.S. land grant university system with contact information.

AESG3:I35/Institution	Crop/Genus/Species	Contact
Clemson University	Specialty Capsicum	Dr. Sandra Branham; [email protected]
Clemson University	Brassica spp. greens	Dr. Sandra Branham; [email protected]
Colorado State University	Specialty Capsicum	Michael Bartolo; [email protected]
Colorado State University	Specialty vegetables	Dr. Mark Uchanski; [email protected]
Cornell University	Specialty Capsicum	Dr. Michael Mazourek; [email protected]
Michigan State University	Fragaria spp.	Dr. Cholani Weebadde; [email protected]
Michigan State University	Stevia rebaudiana	Dr. Ryan Warner; [email protected]
New Mexico State University	Specialty Capsicum	Dr. Dennis Nicuh Lozada; [email protected]
North Carolina State University	Stevia rebaudiana	Dr. Todd Wehner; [email protected]
North Carolina State University	Vaccinium spp.	Dr. Hamid Ashrafi; [email protected]
Ohio State University	Specialty Capsicum	Dr. Leah McHale; [email protected]
Oregon State University	Hazelnut	Dr. Shawn Mehlenbacher; [email protected]
Oregon State University	Mint, brambles	Dr. Kelly Vining; [email protected]
Oregon State University	Specialty Capsicum	Dr. James Myers; [email protected]
Purdue University	Brassica spp. greens	Dr. Jules Janick; [email protected]
Rutgers University	Specialty Capsicum	Dr. James Simon; [email protected]
Rutgers University	Specialty vegetables	Dr. James Simon; [email protected]
Rutgers University	Vaccinium spp.	Dr. Nicholi Vorsa; [email protected]
Texas A&M University	Specialty Capsicum	Dr. Kevin Crosby; [email protected]
University of Arkansas	Leafy greens	Dr. Ainong Shi; [email protected]
University of California Davis	Specialty Capsicum	Dr. Allen Van Deynze; [email protected]
University of California Davis	Fragaria spp.	Dr. Stephen Knapp; [email protected]
University of California Davis	Nut crops	Dr. Patrick Brown; [email protected]
University of California Davis	Nut crops	Dr. Thomas Gradziel; [email protected]
University of California Davis	Nut crops	Dr. David Neale; [email protected]
University of California Davis	Nut crops	Dr. Dan Parfitt; [email protected]
University of California Davis	Specialty vegetables	Dr. Lynn Epstein; [email protected]
University of California Riverside	Specialty vegetables	Dr. Mikeal Roose; [email protected]
University of Florida	Bramble	Dr. Zhanao Deng; [email protected]
University of Florida	Specialty Capsicum	Dr. Bala Rathinasabapathi; [email protected]
University of Florida	Fragaria spp.	Dr. Seonghee Lee; [email protected]
University of Florida	Vaccinium spp.	Dr. Patricio Munoz; [email protected]
University of Florida	Vanilla planifolia	Dr. Alan Chambers; [email protected]
University of Hawaii	Tropical fruits	Dr. Richard Manshardt; [email protected]
University of Minnesota	Specialty vegetables	Dr. Tom Michaels; [email protected]
University of Puerto Rico	Specialty Capsicum	Dr. Linda Wessel-Beaver; [email protected]
University of Wisconsin	Organic produce	Dr. Julie Dawson; [email protected]
University of Wisconsin	Specialty vegetables	Dr. Irwin Goldman; [email protected]
University of Wisconsin	Vaccinium spp.	Dr. Juan Zapala; [email protected]
USDA/ARS	Specialty vegetables	Dr. Philipp Simon; [email protected]
USDA/ARS	Solanaceae	Dr. John Bamberg; [email protected]
USDA/ARS	Specialty Capsicum	Dr. John Stommel; [email protected]

. The trends reflect the factors described in the previous paragraph. This summary is not complete since it was compiled from on-line sources that are often fragmentary and difficult to standardize. Not surprisingly, most plant breeding of specialty crops takes place in states with diverse agricultural economies such as California and Florida. There are, however, significant projects in states with more modest economies such as Colorado, Michigan, Oregon, South Carolina, and Wisconsin. No doubt the trend towards greater diversity in the range of species under development will expand to include more states and crops within states in the future.

Breeding programs in specialty horticultural food crops in concert with demand for new cultivars in the private sector will spawn more activity by USDA/ARS/NGS to actively collect, maintain, and evaluate germplasm of these species. Plant breeders are an important primary clientele for this collection and others like it worldwide.

3. Specialty Crops Germplasm Maintained by the USDA/ARS/NGS

The GRIN system is an important source of new genes for plant breeders to address problems facing growers of the crop species they are working on. Traveling to the Centers of Origin and/or Diversity of the species is increasingly impractical and wrought with political difficulties as germplasm is viewed progressively as a sovereign resource rather than one of universal access. Mining germplasm is also expensive, time-consuming, and dangerous.

The process of germplasm acquisition has changed as political forces intervened during the mid-20th century. In 2021 it is more likely that new accessions are contributed to the NGS collection through mutual agreements between countries or plant breeders to share resources equitably. It makes more sense for local plant explorers to collect potentially valuable genetic resources in their own jurisdictions than to send in a team of foreign scientists to appropriate seeds and other materials from natural habitats.

The NGS plant collection currently includes 601,116 different accessions of 16,289 species, including virtually all the ethnic and specialty crops of interest to United States (U.S.) agriculture. The overwhelming majority of accessions consist of under ten different crop species. While collections of other “minor” species are expanding progress is slow due the political forces described earlier. Demand for germplasm of a specialty horticultural crop species in the U.S. does not necessarily coincide with that in the country/countries that encompass the Centers of Origin and Genetic Diversity for that species.

The NGS accessions are assigned with different subunits of the organization that are dispersed geographically. Plant Introduction Stations and Germplasm Repositories tend to specialize in crop species according to common handling and growing methods. In some cases, a given species may be housed at more than one subunit of NGS. The entire collection is duplicated at the National Laboratory for Genetic Resource Preservation in Fort Collins, CO. Researchers have access to collections through the GRIN database from the PI Stations and Repositories but not the NLGSP. Geographic dispersion of the collections is an important strategy to guard against genetic losses due to catastrophes.

A summary of the NGS collections considered to be specialty horticultural food crop species is provided in

Table 3. A partial list of collections of seeds and vegetative propagules of plant species considered to in this paper to include specialty horticultural food crops maintained at USDA/NGS Plant Introduction Stations and Germplasm Repositories.

Genebank Location	Crops and Species	No. of Accessions	Crops and Species	No. of Accessions
Ames, IA, USA	Actaea	43	medicinals	1084
	Apiaceae	2832	mint	170
	Brassica	2019	Ocimum	106
	Calendula	78	Origanum	21
	Cichorium	285	Pastinaca	59
	Cucumis	4943	Perilla	25
	Cuphea	638	Portulaca	13
	Jerusalem artichoke	53	Potentilla	105
	Lamiaceae	178	Prunella	72
	Malvaceae	3	spinach	413
Corvallis, OR, USA	blue honeysuckle	16	pawpaw	15
	elderberry	84	quince	161
	goji berry	3	Ribes	429
	hazelnut	504	Rubus	571
	hops	2	Fragaria	1975
	medlar	32	Vaccinium	641
	mint	304	wintergreen	3
	mountain mint	51
Davis, CA, USA	almond	323	olive	142
	apricot	298	persimmon	69
	fig	149	pistachio	185
	hardy kiwifruit	39	plum	171
	kiwifruit	92	pomegranate	179
	mulberry	46	walnut	386
Geneva, NY, USA	Allium	784	cabbage	811
	Apium	159	radish	702
	asparagus	29	squash	506
	Brassica	3043	tomatillo	99
Griffin, GA, USA	Bambara	30	peanut	8208
	Capsicum	4829	Cucurbitaceae	1462
	eggplant	490	gourd	655
	hibiscus	473	legume	1177
	okra	1734	sesame	1141
Hilo, HI, USA	breadfruit	57	macademia	24
	carambola	38	papaya	24
	guava	44	peach palm	15
	litchi	93	pili nut	5
	Longan	18	Rambutan	44
Miami, FL, USA	Acerola	5	Annona	18
	avocado	156	jackfruit	10
	Caimito	3	litchi	20
	Canistel	12	mango	321
	Carambola	12	Sapodilla	51
	coconut	13	tamarind	6
	custard apple	10	white sapote	3
	Jaboticaba	7
Mayaguez, PR, USA	Cacao	219	Musa	47
	mamey sapote	27
Pullman, WA, USA	Allium sativa	131	lentil	3021
	Allium wild	211	lupin	790
	Astragalus	813	Medic	4776
	fababean	546	rhubard	70
	Lathyrus	582

.

4. Uses of Germplasm in Specialty Crops Breeding and Research: Capsicum Example

Germplasm is of immense importance for plant breeders to address problems of concern to growers and new market opportunities. Examples of issues that affect growers are diseases and arthropod pests and tolerance to environmental stresses such as temperature (cold or hot), drought, and soil salinity. An example of a need driven by a market opportunity is fruit or tissue quality for enhanced flavor or nutrition. In some instances, germplasm provides new opportunities for plant breeders to become more effective or efficient in the development of new cultivars, for example male sterility for seed production of hybrids.

Each crop species presents a unique set of challenges that is often repeated in different crops. For this reason, the present paper will focus on examples of the uses of germplasm in one subset of specialty horticultural food crop species, chili peppers (cultivated species Capsicum annuum, C. frutescens, C. chinense, C. baccatum, and C. pubescens [10]).

A wide germplasm collection of 307 accessions retrieved from 48 world regions and belonging to nine Capsicum species was characterized for 54 plant, leaf, flower, and fruit traits. This represents the first high-throughput phenotyping effort in Capsicum spp. aimed at broadening the knowledge of the diversity of domesticated and wild peppers [11].

4.1. Breeding for Disease Resistance

The dynamics of host–pathogen interactions are in constant flux for all plant species. New pathogens and races of existing pathogens appear over time and often without warning. Cultivated plants that are the cornerstone of industries that are vulnerable to these forces and depend on the availability of prophylactic chemicals and genetic host resistance to remain viable. With agricultural chemicals under increasing scrutiny for environmental impacts, breeding for genetic resistance to diseases caused by pathogens is progressively more attractive [12,13].

Germplasm repositories at both the World Vegetable Research Center in Taiwan and the USDA/ARS/NGS collection possess number of genotypes found to contain genes for resistance to insect pests, pathogenic nematodes, and fungal, bacterial, and viral pathogens [12].

Examples of new sources of genetic disease resistance discovered in collections of germplasm abound in Capsicum sp. Phytophthora capsici is an important soil-borne pathogen of all Capsicum species that is difficult to control with fungicides, making disease resistance breeding of crucial importance. A large panel of Capsicum accessions was screened for resistance to P. capsici and genes were identified that conferred both strong and intermediate levels [14]. P. capsici has also mutated into several different races based on differences in resistance in host resistance. Capsicum germplasm was employed by Jiang et al. [15] to assemble an effective host differential panel to distinguish races of P. capsici. Mallard et al. [16] screened a panel of Capsicum germplasm accessions for resistance to several isolates of P. capsici and identified a QTL (Pc5.1) that is useful for marker-assisted breeding of resistant cultivars.

Verticillium wilt, caused by the soil-borne pathogen Verticilllium dahliae, is also difficult to control with fungicides. As with P. capsici, genetic resistance in cultivars is crucial for growers to avert this serious disease. Fortunately, evaluation of a large panel of Capsicum accessions revealed several sources of resistance that has been used by plant breeders [17]. C. baccatum is known to be an excellent source of genes for resistance to the powdery mildew (Leveillula taurica) and anthracnose (Colletotrichum sp.) pathogens of cultivated peppers. Lee et al. [18] used a hybrid of C. baccatum accessions to map the locations of the genes underlying resistance to these disease pathogens.

The bacterial spot disease caused by pathogenic Xanthomonas species causes significant crop damage on tomato and pepper in tropical and subtropical regions throughout the world. Host resistance has been one of the key components of integrated disease management approaches to mitigate bacterial spot. Genome-wide association study (GWAS) on a diverse USDA/ARS/NGS collection of pepper germplasm revealed several sources of genetic resistance to a highly pathogenic isolate causing bacterial spot, X. gardneri [19].

Host resistance to viral pathogens is desirable because they are usually spread by arthropod vectors. Sources of resistance to tobacco mosaic virus, Cucumber mosaic virus, Pepper veinal mottle virus, Chilli veinal mottle virus, and Peanut bud necrosis virus were identified and characterized from screenings of the World Vegetable Center Capsicum germplasm collection [12]. Resistance to pepper mild mottle virus has also been found in Capsicum germplasm collections [20]. Pepper huasteco yellow vein virus (PHYVV) is the main virus of Capsicum crop species in Mexico and Central America and can cause damage in northern latitudes as well. Sources of genetic resistance to PHYVV were discovered within germplasm of C. annuum [21].

Pathogenic nematodes such as root knot (Meloidogyne incognita) are a serious crop production challenge for specialty Capsicum growers in certain parts of the world. Selections with genetic resistance to root knot nematodes were identified in panels of Capsicum germplasm accessions [22]. These selections were useful especially as rootstocks. Gisbert et al. [23] used a different set of accessions to find resistance in Capsicum to M. incognita.

4.2. Breeding for Fruit Quality

The breeding of crop cultivars for consumer demands and value-added differentiation can enhance profits to growers and food processors, including Capsicum. Example of value-added consumer traits in chili peppers are fruit flavor and nutritional quality. Capsicum germplasm collections have been explored extensively for genes that enhance flavor, nutritional and color [24].

Several studies have been conducted of the variation of basic architecture of pepper fruits. Mature fruit of 330 accessions of C. chinense from the USDA/ARS/NGS Capsicum germplasm collection were characterized for fruit length, width, weight, and color. These data define the variability for mature fruit characteristics within this germplasm collection and provide a baseline against which future introductions/acquisitions can be compared [25]. Stommel et al. [26] evaluated and selected fresh-cut attributes in pepper accessions with diverse fruit phenotype selected from available cultivars and the USDA, ARS Capsicum genebank. In another study, a total of 18 traditional accessions were compared with five hybrids and two ecotypes with similar fruit shape/size were used to identify genes that impact fruit flavor and nutritional quality [26].

Another crop attribute of importance in Capsicum is fruit color, with implications for both aesthetic and nutritional quality. Combinations of pepper accession genomes were employed to elucidate the underlying genotypes of orange chili peppers [27]. Allelic variations were found in six carotenoid biosynthetic genes, including phytoene synthase (PSY1, PSY2), lycopene β-cyclase, β-carotene hydroxylase, zeaxanthin epoxidase and capsanthin-capsorubin synthase (CCS) genes, in 94 pepper accessions by single-molecule real-time (SMRT) sequencing [28]. More recently, Guzman et al. [29] investigated the diversity of carotenoids and phenolics in germplasm from three Capsicum (chili pepper) species, C. annuum, C. baccatum, and C. chinense. Results indicated that in only nine of 31 Capsicum accessions, lutein represented at least 50% of the total carotenoid amounts in each accession.

Several studies have employed Capsicum germplasm resources to study the expression of genes that modulate anthocyanin pigment expression in fruit. Stommel et al. [30] reported on the discovery and sequencing of transcription factors in Capsicum annuum from a study of a range of different phenotypic backgrounds. In other studies by this group, Capsicum germplasm resources were also used to discern the interactions of anthocyanins and carotenoids in pepper fruit pigmentation [31].

Many researchers have explored levels of fruit pungency present within collections of Capsicum germplasm. Ninety Capsicum accessions, including C. chinense, C. frutescens, C. baccatum, C. annuum, and C. pubescens) selected from the USDA Capsicum germplasm collection were screened for their capsaicinoids content using gas chromatography with nitrogen phosphorus detection (GC/NPD) [32]. Antonious et al. [33] selected candidate accessions of hot pepper having high concentrations of ascorbic acid, capsaicin, free sugars, and total phenols for use as parents in breeding for these compounds. The great variability within and among Capsicum species for these phytochemicals suggests that these selected accessions may be useful as parents in hybridization programs to produce fruits with value-added traits. More recently, a study demonstrated the use of tools for predicting the spiciness of fresh peppers useful for breeding programs and consumers based on levels of volatile organic compounds (VOCs; [34]).

Many studies have focused on biochemical components of pathways that culminate in enhanced levels of compounds that promote human nutrition. For example, Rodriguez-Burruezo et al. [35] presented the results of studies on the ascorbic acid content (AAC) and total phenolics (TPs) of eight important Spanish pepper landraces. Genotype × environment interaction enabled the identification of germplasm accessions with high flavonoid content grown under organic conditions at both ripening stages, particularly total flavonoids and luteolin at the fully ripe stage [36].

Molecular markers linked to QTLs controlling post-harvest fruit water loss in pepper may be utilized to accelerate breeding for improved shelf life and inhibit over-ripening before harvest. Genetic materials and molecular markers developed in this study may be utilized to breed peppers with improved shelf life and inhibited over-ripening before harvest [37].

4.3. Breeding for Male Sterility and Floral Development

Flower and fruit development are important for breeding of successful Capsicum cultivars. The use of germplasm to study floral development, morphology, and physiology in Capsicum was clearly demonstrated [38]. Male sterility is potentially useful to reduce the cost of hybrid pepper seeds. Markers linked to nuclear restorer genes were identified to facilitate breeding for NMS and CMS. Information on both NMS and CMS systems in pepper was reported and the prospects for their use in breeding for heterosis discussed [39]. Screening of C. annuum and C. baccatum accessions led to the discovery of new sources of Rf genes for male sterility [40].

4.4. Breeding for Salt and Drought Tolerance

A few efforts have been mounted to assess levels of drought and salt tolerance present in collections of Capsicum germplasm. For example, 20 germplasm accessions of chili peppers were evaluated for salinity tolerance. ‘Early Jalapeno’ and ‘AZ-20’ were relatively tolerant to salinity among the tested genotypes, whereas ‘TAM Mild Habanero’ and ‘Ben Villalon’ were sensitive [41].

4.5. Germplasm Collection Strategies and Maintenance

The availability of enormous genetic variability in Capsicum germplasm collections has been utilized to elucidate strategies for the efficient and effective collection, evaluation, and maintenance of plant genetic resources. In one such study, various strategies for slowing or reversing the loss of genetic variation from Capsicum germplasm collections were proposed and discussed by Gonzalez and Bosland [24]. Many studies on the use of traditional phenotypes, molecular markers and other emerging technologies such as GIS to assess genetic variability in Capsicum germplasm collections as compared to wild and domesticated populations have been conducted since then. For example, Toquica et al. [42] used AFLPs to quantify genetic variability in Capsicum collections from the Columbian Amazon basin. They identified genetic clusters that accounted for most of the variation observed and concluded that current germplasm collections have captured most extant diversity. Later, Brilhante et al. [43] described the use of qualitative and quantitative descriptors and ISSR markers to characterize 69 pepper accessions of four Capsicum species from different regions of Brazil. In another study, morphological measures of genetic variability in Chilean Capsicum germplasm showed that the values of central tendency and dispersion showed variation coefficients greater than 25% for fruit quantitative descriptors [44].

GIS was employed to study distribution of the wild species C. flexuosum in Paraguay [45]. The derived information will be helpful in devising new methods for efficient ex situ germplasm conservation. Later, Albrecht et al. [46] conducted a combined diversity analysis on the USDA-ARS Capsicum baccatum germplasm collection using data from GIS, morphological traits, and DNA fingerprints. They concluded that division of the domesticated C. baccatum germplasm into two major regional groups (Western and Eastern) was supported by the pattern of spatial population structures. Further analyses by this group of inter-specific relationships across selected Capsicum species supported independent lineages for the two crossability groups within Capsicum, the baccatum species-complex (including C. baccatum) and the annuum species-complex (including C. annuum, C. chinense, and C. frutescens). These results will be useful for identifying accessions for crop improvement and guiding the development of in situ and ex situ conservation programs [47].

Another study used multivariate techniques with both qualitative and quantitative descriptors in the five domesticated species of Capsicum for grouping them after assessing inter- and intra-specific variation [48]. Characterization of bell pepper germplasm provided valuable information for strengthening breeding programs through the selection of parents based purely on morphological characters [49].

An exploration and collection mission for wild populations of Capsicum was carried out in the fall of 2006 and 2007, in 13 Mexican states and in the U.S. states of Arizona and Texas. The aim was to expand the number of accessions of wild chili pepper (Capsicum annuum var. glabriusculum and Capsicum frutescens) that are publicly available for research in plant improvement and for subsequent use in an inquiry into the domestication of C. annuum [50]. Other groups explored methods to improve the efficiency of germplasm maintenance by eliminating redundancies. For example, computer software was used to select 28 representative Capsicum accessions to form a core collection, which maintained a similar level of diversity to that of the overall 230 Capsicum accessions [51].

Settle nesting of core-collections of 8, 16, 32, 64 and 128 C. annuum accessions was found to capture from 37 to 90% of the genetic diversity for further sequencing efforts and establishment of high throughput genotyping assays. By compiling phenotypic and genotypic data, a larger core-collection of 332 accessions was established, capturing 97% of the C. annuum genetic and phenotypic diversity for further genetic association studies [52]. In another such study, a total of 240 accessions were selected from 3821 Capsicum accessions based on transcriptome-based 48 SNP markers with genome-wide distribution and 32 traits using a systematic approach [53]. They identified four basic clusters (A, B, C, and D) and concluded that core germplasm collections with a minimum number of accessions and maximum genetic diversity of cultivated species and wild relatives will facilitate easy access to genetic material as well as the use of hidden genetic diversity. Among the 11 species examined in this study, Capsicum annuum showed the highest genetic diversity, whereas the wild species C. galapagoense showed the lowest genetic diversity. Most of the accessions from European countries are distributed in the A and B groups, whereas the accessions from Asian countries are mainly distributed in C and D groups. A total of 240 accessions were selected from 3821 Capsicum accessions based on transcriptome-based 48 SNP markers with genome-wide distribution and 32 traits using a systematic approach.

More recently, genetic polymorphism, cross transferability (CT), and genetic diversity were examined among the 54 different accessions of Capsicum species including 49 of C. annuum, three of C. baccatum and two of C. frutescens, using a set of 36 start codon targeted (SCoT) primers. Results showed the effectiveness of SCoT marker system for characterizing and assessing genetic diversity of Capsicum germplasm [54].

4.6. Genomic Structure and Evolution

By analyzing the dynamics of mutations and genomic fluctuations over evolutionary time in Capsicum germplasm collections, important information on crop domestication has been obtained. For example, a broad spectrum of domesticated and wild C. baccatum germplasm and AFLP markers were used to describe the species’ molecular diversity and population structure in the South American gene pool. Results suggested that C. baccatum was domesticated in multiple sites and that its evolution took two lineages followed by lineage differentiation. Results of clustering analysis also suggested that C. baccatum likely originated from present day Paraguay [47]. Genetic diversity of Chinese pepper landraces was concluded to be structured by migration followed by human selection for cultivar types in agreement with consumption modes and adaptation to the highly diversified agro-climatic conditions [55].

Traditional cytogenetic methods showed that there are two different evolutionary lines in the genus and that the native south-eastern Brazilian species belong to the ancestral Capsicum gene pool [56].

Germplasm collections, including Capsicum, have been instrumental in gene mapping and genomics framework studies. For example, Nimmakayala et al. [57] aimed to resolve the genetic diversity and relatedness of C. annuum germplasm by use of simple sequence repeat (SSR) loci across all chromosomes. They identified three major quantitative trait loci located on C. annuum chromosomes 8, 9, and 10 that were associated with fruit weight. In another study, the genetic diversity of 1904 accessions of pepper conserved at the National Mid-term Genebank for Vegetables, Beijing, China, was analyzed based on 29 simple sequence repeat (SSR) markers, which were evenly distributed over 12 pepper chromosomes [58]. The availability of saturated linkage maps will great enhance breeding progress in the future.

5. Conclusions

The Government of the United States of America has established a long-standing culture of preserving a collection of germplasm from all around the world, which serves as a resource for maintaining the country’s rich crop diversity including specialty and ethnic crops that are native or foreign to the country. Currently more than 600,000 different accessions of 16,289 species, including virtually all the ethnic and specialty crops of interest to United States (U.S.) agriculture, are preserved at different locations all around the country. In addition, plant seeds and asexual propagules are maintained at various laboratories and facilities geographically dispersed in the country including the NGS and many public land-grant university institutions. This germplasm repository is of immense significance to the future food security of the country as it becomes more compelling to diversify the crop content of the food system to respond appropriately to the country’s changing demographics and the unfolding realities of climate change. Researchers and the public have access to these resources and are free to use them to support a healthy workforce far into the future.