PLS 221                                                                                       Instructor: C. F. Quiros

Solanacea: PEPPER: Capsicum spp

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Slides


Origin and distribution of the genus

Wild and domesticated species, identification

Horticultural types of C. annuum

Crossing relationships

Cytogenetics: polyploids and aneuploids.

Primary trisomics and gene mapping.

Inheritance of most important traits

Gene mapping and markers

Gene transfer schemes

Disease resistance


Genus Capsicum

The basic chromosome number of the genus is x=12, all the species are diploids, most are 2n=2x=24, including the cultivated ones. A few wild species have 2n=26.

Capsicum originated in the New World, where at least 20 species have been reported.

Seeds found in cave dwellings indicate the natives were enjoying peppers 7000 BC, along with potatoes in the Andes. In Mexico dry pepper fruits and seeds were recovered from 9000 years old burials in Tamaulipas and Tehuacan.  Domestication might have taken place 10,000 to 12,000 years ago.  Therefore, pepper was one of the first plants domesticated in the AmericasChiles were introduced to Europe by Columbus resulting in their almost immediate incorporation to existing recipes. From there, they quickly expanded to Africa and Asia and eventually to North America. In India and China in particular, chiles have become an important component of their cuisines.

 There are 5 domesticated species of Capsicum. Domestication of these species has taken place in two independent centers, 1) Mexico and Guatemala through Colombia on the North, and 2) in the Andes of Peru and Bolivia in the South.

Wild peppers can be distinguished from the cultivated ones by several characteristics:
 

Wild

Cultivated

Small fruit 

Vary in sizes

Red 

Vary in colors

Pungent (c gene)

Sweet or pungent

Soft flesh (deciduous, s gene)

Hard flesh, few soft

Upright fruits (up)

Mostly pendant

Light fruit load

Mostly heavy

Cross pollinated

Mostly selfed (short style)

Domesticated species:

1) C. annuum. Domesticated in the highlands of Mexico and Central America. It is the most common species in North America with a wide range distribution, from Mexico, the main center, to Northern Argentina.

Two botanical varieties are recognized:

var. aviculare:  wild

var. annuum: cultivated

Great variability found for this species, including hot and sweet pepper, for example we found the following types: bell peppers, Anaheims, jalapeños etc.

Most of the cytogenetic information and plant breeding efforts have been focused on this species because of its economic importance.

2) C. frutescens  is mostly a tropical species, tolerant to heat, spreads from the South USA, Yucatan Pen. to Northern Brazil. It was domesticated in a wide area from Colombia to Mexico. An example of a cultivated type is the Tabasco pepper used for the Tabasco sauce.

3) C. chinense  is found mostly in the Amazonian Basin to Bolivia. Probably domesticated in the Bolivian Andes. In the Yucatan peninsula the popular pepper Habanero belongs to this species.

(see the three species side by side) Note the number of flowers per node.

4) C. baccatum. Found in Brazil, Peru, Bolivia Chile and Argentina.

Two subspecies are recognized:

ssp. baccatum. Wild form from S. Ecuador to Bolivia

ssp. pendulum. Cultivated in the Eastern side of the Andes.

Both ssp. are cross fertile.

This species is grown in a limited scale in California for pickling as variety Bruno or mild italian.

5) C. pubescens.  Species endemic of high elevations, cold tolerant, some accessions are self-incompatible. It is found in Colombia, Peru, Ecuador Bolivia. It cannot stand hot climate and has purple flowers and black seeds. This species is cultivated in the highlands from Colombia to Argentina, although it is often found under cultivation in Mexico.

Each of these species are identifiable morphologically by the following traits:
 

Species

Flower color

Number flw/node

Seed color

Calyx constriction

Flower position

annuum

white

1

tan

absent

 

frutescens

greenish

2-5

tan

absent

erect

chinense

White/ greenish

2-5

tan

present

pendant

baccatum

White with yellow spot

1-2

tan

absent

 

pubescens

purple

1-2

black

absent

 

Species range of distribution

 Distribution range for C. annuum (top left), C. baccatum (top right), C. pubescens (bottom left), and C. frutescens/chinense (bottom right)

Although the taxonomic boundaries of Capsicum are still very uncertain, the cultivated species can be arranged into a phylogenetic tree consisting of three main branches. One formed by annuum, chinense and a frutescens, which can cross with each other producing partially fertile hybrids. They form a common gene pool.

Hybrids between any of these three species with C. baccatum are much more difficult to get and the hybrids are highly sterile.

White flowered species:

1) Annuum-chinense-frutescens complex:

C. galapagoense, endemic of the Galapagos Islands.

C. chacoense

2) Baccatum:

C. praetemissum  has yellow spots in corolla also, perhaps derived from C. baccatum ssp baccatum (progenitor of pendulum) by founder effect. Corolla spots are due to a single dominant gene.

Purple flower species:

3) Pubescens:

C. eximium has light color seed, white to lavender corolla and is self-compatible.

C. cardenasii. It is restricted to La Paz, Bolivia, very interesting species with tubular, light purple corolla, brown seed and self-incompatible. Crosses in both directions to C. eximium, producing highly fertile and vigorous hybrids.

Both species cross easily as females to C. pubescens.

C. tovari is a white flowered species, hybridizes to C. eximium and C. baccatum, but does not hybridize to C. pubescens.

4) Other species: C. schottianum, C. bufforum, from SW Brazil and NE Argentina, have black seeds.

There may be still in nature Capsicum species which have not been yet described. For example, in Madre de Dios, Peru, an unknown species with approximately 20 flowers per node and copper color flowers was observed.

Crossing relationships between these species: Determined by interspecific hybridization by reciprocal crosses.
 

Breeding System:

Most of the Capsicum species are self-compatible and facultative inbreeders. Outcrossing varies from 12 to 70%, as in tomatoes the cultivated species have their stigma at the anther level.

Unilateral incompatibility is present in crosses involving the species of the pubescens complex and all other species.

Hybridization Barriers:

A number of hybridization barriers due to failure of fertilization, seed development or embryo growth occur in certain interspecific crosses of Capsicum. These can be classified as follows:

1) Pre-fertilization: inhibition of pollen tube growth.

i.e.. annuum x pubescens.

2) Post-fertilization: endosperm breakdown, resulting in lack of germination.

e.g.  pubescens. x chinense.

3) Post-germination: hybrid lethality.
e.g. baccatum x tovarii,
C. annuum x galapagense, 
chinense x baccatum.
 

Cytogenetics:

It is not as well developed as in tomato, because pachytene is not easy to study in Capsicum species. The karyotypes of the different species are very similar to each other. It has one pair of acrocentrics and the rest of the chromosomes are meta or sub-metacentric.

Reciprocal translocations have played an important role in speciation of Capsicum.

Euploid variation:

Very few pepper triploids and tetraploids have been reported in nature. Haploids, however, are much more common. They show up spontaneously in twin seedlings at the rate of 1/1000.

The pepper haploids of the variety Doux Long des Landes are unusual because of their ability to generate aneuploids, which has resulted in the development of a primary trisomic series in pepper.

Progeny of pepper haploids:
 

Chromosome number

12

24

25

26

27

other

%

0.4

93

2

1

0.6

3

Total progeny 680 plants

11 primary trisomics have been reported in pepper, they have been described by organ colors. Only three have been cytological associated to their respective chromosome. It is difficult to identify them at seedling stage.
   

 

% Transmission

Trisomic

Name

Description

Freq.

self

BC

Triplo-1

violet

Violet anthers

16

4

0

 

indigo

Dark blue ant

14

47

47

 

blue

Blue/green Lvs

20

24

17

 

green

Dark green Lvs

14

32

32

Triplo-11

yellow

Yellowish Lvs

9

21

28

 

orange

Orange/red frt.

8

14

12

 

red

Dark red frt.

6

43

51

Triplo-12

purple

Arbitrary -NOR

2

6

10

 

black

DKr green immature frt.

4

21

33

 

brown

Brown stamen

4

34

22

 

bistro

Arbitrary

2

4

15

Diploid cv. Doux Long des Lands used to derive these trisomics has the following traits: yellow stamens with bluish pigmentation, medium green foliage, red fruits.

Male transmission is zero.

Triplo-12, upon selfing regenerates the whole trisomic series.

Genes located by trisomic analysis:

L1, TMV resistance on trisomic brown

C, presence of capsaicin, on tr 11, yellow

y, yellow fruit in tr indigo

a, anthocyanin in foliage, in tr red

up, upright fruit, tr black

At least 100 mutants reported, but linkage analysis and chromosome assignment rather limited.
 

Genes of importance:

1) Fruit shape, and size. round fruit vs long, determined by dominant O, from accession of C. chinense. However, in most crosses fruit shape and size is polygenic. For fruit size, small is dominant over large. More than 30 genes involved. At least 4 BC needed to recover large size. QTL analysis reveals 6 QTL, at least 2 shared with tomato. Not clear if any of them corresponds to Sun1.  Fruit size, 3QTL locations also shared with tomato.

2) Pungency or heat: Due to family of alkaloids called  capsaicinoids: capsaicin, dihydrocapsaicin and its isomers. They are fat-soluble, odorless, flavorless and colorless compounds. It is measured in Scoville units (dilution factor at whci heat can be still detected by taste). Capsaicin content is determined mainly by gene Pun1 (before C) and a few modifier genes. Pun1 corresponds to a acyltransferase enzyme. Sweet peppers have a 25bp deletion in this gene. (Stewart et al 2005.). Prasad et al 2006 claim it is another gene, csy1, (capsaicin synthase. It is 10cM from Pun1, possible the wrong gene since it expresses in sweet peppers too and was found using Pun1 primers in spite of lack of sequence similarity between both genes( I. Paran pers com)..  6 QTL control amount of capsaicinoids. (Ben-Chaim et al 2006).  Loss of function of aminotransferase pAMT in mutant CH-19 Sweet of C. annuum due to insertion results in loss of pungency. This mutation is unique and not present in other non-pungent types. This mutant produces capsinoids, which are natural analogs of pungent capsaicinoids. Capsinoids have anti-cancer and anti-oxidant activity. (Lang et al 2009)
Flavor is not due to capsaicinoids but to other aromatic compounds.

Hottest peppers found in species C. chinense, ‘Bhut Jolokia’ and habanero peppers (Canto-Flick et al 2008).

3. Fruit color:
Immature: varies from black, purple, passing through green, yellow and white.

At least 5 loci account for these colors. Purple color in fruit and other organs is determined by gene A, on chromosome 10. The intensity of color is modulated by modiifier by MoA. A is linked to O at 6.5cM, which is responsible for round fruit shape. Interestingly, potato displays a similar linkage between tuber shape and anthocyanin. (Chaim et al. 106:889-894,2003).

Mature: These include many colors due to the actions of four major genes in the carotenoid pathway. These genes were reported by Hurtado and Smith (1985) through genetic analysis. See also Thorup et al. (PNAS 97:11192-7, 2000)

y :  yellow

cl : chlorophyll retention

c1 : reduces pigments by 1/10

c2 : reduces pigment to traces.
 

Color genotypes:

y cl c1 c2

+  +  +  + red

+  + c1 + light red

+  +  + c2 orange

+  + c1 c2 pale orange

+  cl +  + chocolate

y  +  +  + yellow

y  + c1 + pale yellow

y  + + c2 lemon yellow

y  + c1 c2 white

y  cl  -  - green
 

More recent studies on fruit color genes: Lefebvre et al. (1998), Popovsky and Paran (2000) ,(Huh et al. 2001), Kang et al. (2001).

4. Male sterility: (see review by Shifriss, 1997)

Nuclear male sterility: At least 12 genes reported, obtained mostly by mutagenesis. Seems to be always the same gene.

ms-509 is used in France for hybrid seed production. A SCAR marker for ms reported by Lee et al (2009).

 

Cytoplasmic male sterility was found by Peterson in an introduction of C. annuum from India (PI164835). It is determined by nuclear gene ms interacting with S cytoplasm. Restorer Ms alleles are found mostly in hot pepper accessions but must be selected for stability.

Markers for cms and fertile plants available, rf in S cytoplasm, restorer Rf.  C. annuum (Guyas et al 2006, Jo 2010)

Cytoplasmic male sterility have also been reported by interspecific hybridization.

C. baccatum x C. annuum. No adequate fertility restoration have been found for this system.

C. annuum (male) with C. chacoense (female).
No restorers (N/SMsms) or maintainers (Nmsms) have been found.

5. Isozymes and DNA based markers
Isozyme loci were reported first by McLeod et al. (1983), mostly to study genetic variation and distances among different Capsicum species. A limited number of accessions was used for the study and practically no genetic analysis was reported.

Tanksley (1984) reported the inheritance of 14 isozyme loci, their linkage and location on their chromosomes by trisomic analysis in an interspecific cross of C. annuum x C. chinense. This work has now expanded to include a large number of RFLP and other DNA-based markers.

Tanksley and Bernatsky (1988) used RFLP's for comparing tomato cDNA probes on pepper, and a few cDNA pepper leaf clones. Although both species have the same chromosome number, the DNA content of both species is different, tomato has only 1.0 pg/haploid nuclei, while pepper has 2.76 (potato has also approximately 1pg). Based on a total of approximately 100 loci marking 11 of the 12 chromosomes, they reached the following conclusions:

Gene repertoire is conserved between both species. Extensive chromosomal rearrangement, only a few conserved sequences have remained.

Prince and Tanklsey (1993), and Lefevre et al. (1995) have expanded this work. Lefebvre et al. (1995) constructed an intraspecific map in C. annuum with RFLP and RAPDs in F1 doubled haploids. On the basis of this work, sixteen chromosome break events could be postulated differentiating the pepper from the tomato maps.

Livingstone et al. (1999) construted and interspecific map in the F2 of  C. annuum x C. chinense covering 1246 cM in 11 large and 2 small linkage groups. The higher resolution of this map disclosed additional chromosome breaks occurring during the divergence of both species.

Kang et al. (2001) produce a map by crossing C. annuum and C. chinense (Habanero). The map is based mostly on AFLPs and RFLPs based on cDNAs from pepper. These includes genes know to be involved in carotenoid and capsaicin biosynthesis.

A more cent and integrated map havw been published recently by Paran et al (2004). Bocrosatellite loci have been reported by Lee et al. (2004).

Although pepper has 3 fold greater DNA content that tomato, the two species has equivalent genic content. Both genomes conserved linkage blocks.

Marker applications:

1. Isozyme markers were used by Tanksley and Iglesias (1984) to determine the number of QTLs controlling multiple flower character in C. chinense.

2. An alternative method to cut the number of BC when transferring a desirable trait has been proposed by Daskalov (1984).

3. RFLP markers were used by Prince et al. (1992) to determine genetic distances among Mexican accessions of C. annuum, and C. frutescens.


4. Gene mapping and tagging. In addition to the genes involved in carotene synthesis, two loci conferring resistance to the pepper mottled potyvirus Pvr4, Pvr7 have been found tightly linked on chromosome 10. These are also linked to Tsw, conferring resistance to tomato spotted wilt virus.

 

QTLs reported for disease resistance genes (Caranta et al. 2002, Pflieger et al 2001), fruit related traits (Blum et al. 2003, Chaim et al 2001), yield (Rao et al, 2003a).

 

BAC library available for genomics research (Yoo et al. 2003).

Tissue culture and transgenic plants:

Androgenesis: In addition to parthenogenesis in the form of twin seedlings, haploids can be also obtained in pepper by anther culture. Dolcet-Sanjuan et al (1997) reported an improved protocol for androgenesis.

These have been used in pepper breeding for resistance to tobacco mosaic virus, potato Y virus, nematodes and Phytophtora.

Also androgenesis has been used in interspecific hybridization, to scale down ploidy of amphidiploids between species that give sterile diploid hybrids. 1) Select for amphidiploids of higher fertility to increase the chance of genomic recombination between the chromosomes of both parental species. 2) Anther culture of amphidiploid fertile hybrids 3) Screen dihaploids for desirable recombinants. 4) Perform backcrosses to species used as recurrent parent, to eliminate undesirable genes.

Transformation techniques: In pepper these have lagged behind other crops because of the lack of efficient regeneration techniques. Somatic embryogenesis from zygotic embryos has been reported as an alternative to organogenesis in C. annuum (Binzel et al. 1996).

Monoharan et al. (1998) reported transformation of C. annuum with Agrobacterium. Bt pepper resistant to budworm reported by Kim et al. (2002).

Virus resistance lines obtained by Lee et al 2004

Kothari et al 2009 has produced a review on the problems and advances made in Capsicum tissue culture and transformation.

Diseases and virus resistance: A good account on this subject is given in the chapter by Greenleaf (1986), including inoculation techniques.

TMV Gene L, multiple alleles

CMV gene cm plus 7 QTL

Potyvirus: Various resistance genes, MAS for ptv-1

TSWS (tomato spotted wilt topovirus) Tsw, MAS available.

Other diseases: Bacterial spot (Xanthomonas) 8-9 genes, MAS available.

Phytophtora, Psf and Pfr, plus 6 QTL.

Anthracnosis, Anr gene family

Oidium Lmr gene family and 7 QTL.

Nematodes: gene N, for Meloidogyne species. 6 resistance genes found on chromosome 9 in addition to N. MAS available for this trait.

Outlook:

There are lots to be done in Capsicum, including exploration of wild of alternative cultivated species. Most of the latter are exclusively cultivated outside the US.

Basic work is needed for breaking of interspecific barriers. Extensive research is also necessary to solve the virus problem. Transgenic technique needs to be improved for higher efficiency and adapted to use to all five cultivated species. Transgenic approaches may be useful to engineer virus resistance among other diseases. Isolation of major genes involved in secondary metabolites and pigments promises application in the development of functional foods.



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Last modified, April 2, 2010

© Carlos F Quiros, 1998