PLS221                                              Instructor: Carlos F. Quiros

Liliaceae: Asparagus officinalis, asparagus

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Asparagus

Origin, distribution and domestication

Sex determination

Cytogenetics: location of sex chromosome by trisomic analysis

Variety development: kinds of varieties, role of in vitro techniques.

Supermales

Complexity of asparagus breeding

Important traits and their evaluation

Test cycle and correlations between fern and spear seasons

Diseases

Molecular markers, and their applications


Genus

Asparagus, Asparagus officinalis var. altitis. The cultivated species is diploid with 2n=2x=20, DNA content ~1.40pg/1C (1308Mbp). One of the smallest in the family.

Origin and domestication: The wild form A. officinalis var. prostratus, is found in the coast and sandy areas of Eastern Mediterranean Europe, Eastern Asia and South Africa. This species has been used as a medicinal plant and food crop since early civilization by Greeks and Romans. It was introduced to the US by European immigrants. In Europe it is produced as white asparagus, harvesting bleached spears. In the US it is produced as green asparagus, harvesting the green spears after emerging from the soil.

Related species: There are more than 150 species in the genus, 5 of which are closely related species. Some species are grown as ornamentals and for medicinal purposes.

European species:

A. maritimus, is the possible ancestral species to asparagus. Hexaploid. Resistant to salinity. 4x

A. acutifolius, is a spiny wild species, eaten in the Mediterranean by rural people. Drought resistant. 4x, and 2x but probably a different species (Falavigna)

A. tenuifolius Closest relative to A. officinalis. Resistant to acid soils and found at sub-alpine, higher altitude regions.

The other wild relatives are native to South Africa and have perfect flowers. The genome size of these species is half of that of the European species.

Practically nothing has been done on interspecific hybridization. Recent work by Falavigna, crosses 4x officinalis to matritimus/acutifolis hybrid, then reduce back to 2n by anther culture. Maritimus necessary as a bridge species. Purpose is to increase variability in officinalis and transfer disease resistance.

Asparagus is a dioecious, perennial, and bee pollinated.

Sex determination and expression:

Being dioecious, sex determination is of the XX, XY type. However, no heteromorphic X or Y chromosomes can be detected, so sex may be determined by a single locus. The karyotype consists of 6 large and 4 small pairs of chromosomes.

Symbols used for sex genotypes:

XY male (Mm)

XX female (mm)

YY supermale (MM)

Significance of andromonoecy:

Approximately 1/1000 of XY males are andromonoecious (plants with male and perfect flowers). The perfect flowers of these plants develop small fruits with viable seeds, which will produce progeny segregating 3:1 [male (2XY: 1YY), to female (XX)]. The YY (supermales) indistinguishable from the XY males.

Andromonecious males are selected on the basis of berry number.

They should not produce more than 10 per year, otherwise male hybrids will have too many berries thus having the same problem of the females defeating the purpose. That is, female plants tend to be lower yielders, because they spend more energy than males in berry formation. If the berries are removed in the female plants, yields increases 10-20%.

YY supermales can be used to cross with XX for producing all male hybrid varieties as explained below.

Model for control of andromonecious phenotype:

A model has been proposed to explain the genetic basis of this trait, based on two linked loci:

1) Dominant suppressor of female flowers (S)

2) Dominant activator of male flowers (A)

Both genes are closely linked.

Male SsAa

Female ssaa

Rare recombinants, Ssaa, ssAA results in andromonoecious.

Environment also plays a role in the expression of andromonoecy. For example, at the end of the season, males tend to produce a few perfect flowers.

The same structural genes are present in male and female flowers, but different regulatory genes controlling the appearance of reproductive organs.

The pathway for flower development is remarkably similar in both sexes up to meiosis. Until this stage, male and female flowers contain primordia for stamens and pistils. Soon thereafter, the stamens degenerate and pistils develop into female flowers. In male plants, pistils stop growing but do not degenerate, and stamens continue to develop. Sex specific mRNA has been isolated. Also specific protein spots have been identified as sex specific. It seems to be that the absence of translational control in the male rudimentary ovaries causes of inability to proceed with normal ovary development.

Polyembryony:

Occurs in about 1% to 3.5% of the seeds, producing mostly twins, seedlings, although triplets and quadruplets also occur.

These are a source of haploids, triploids and even trisomics. The frequency of haploids is 18/1000 polyembryonic seeds.

Cytogenetics:

Identification of the sex chromosome by trisomic analysis: Trisomics were obtained by Loptier, in Germany by 4x-2x crosses. He used Giemsa stain banding for chromosome identification. After inducing tetraploids, he crossed diploid with tetraploid asparagus, which is only possible using the 4n stock as female. Reciprocal crosses did not produce viable seed.

The resulting triploids XXY and XXX, segregating 1:1, he crossed them to XX and XY plants, respectively.

Triploid offspring of 4x x 2x:   XXXX x XY -->XXY: XXX (1male:1female)

Crosses of triploids x diploids to extract trisomics is done reciprocally. This approach is possible is asparagus.

   Percent of individuals for each of the chromosome number resulting from the 3n x 2n crosses.
 

Chromosome number

XXX x XY

XX x XXY

10

0

0.4

20

0

19

21(trisomics)

28

40

22

30

29

23-24

29

12

25-27

15

0

50

2

0

____________________________________________________________

A total of approximately 300 plants were surveyed in the study.

Unlike trisomics of other species, in asparagus the extra chromosome transmits both in pollen and eggs. However, higher transmission rates was observed for the eggs. Further, the larger chromosomes have lower pollen transmission than the smaller ones, which critical for identifying trisomic ratios. This is because it is not possible to distinguish trisomics from diploids by phenotype, so the offspring from 2n x 2n+1 crosses cannot be sorted in diploid and trisomic groups. Furthermore, trisomics for different chromosomes cannot be distinguished by phenotypic features, so they had to be identified cytologically by Giemsa banding.

Nine of the 10 primary trisomics were recovered. Trisomics for autosome L1 were not obtained. Each chromosome has been named according to their length L1, L2, to L5 (long), M1(medium), S1 to S4 (short).

The trisomic individuals were identified cytologically in the progeny, selecting for male trisomics, hoping that some of them would be trisomic for the sex chromosome, of constitution XXY. Female trisomics were discarded, since those of XXX constitution are not informative because they will not segregate for sex chromosomes. Therefore, they will not serve to detect deviant segregation ratios for sex which is expected only for XXY individuals when compared to expected ratios for XY individuals.

A series of 2n x 2n + 1 crosses were made, using male trisomics were used as pollen parents. The sex ratio of each progeny was studied to identify the trisomic involving the sex chromosome. Ratios deviating from 1 male: 1 female identified the trisomic for the sex chromosome.

Critical ratio:          XX  x  XXY (trisomic for sex chromosomes)

               male gametes

female 
gamete

2X

1Y

1XX

2XY

X

2XX

1XY

1XXX

2XXY

Expected sex ratio for 100% transmission rate of extra chromosome
1 male : 1 female

Therefore, high transmission of the supernumerary sex chromosome in pollen will result in a male: female ratio approaching 1:1, which is the expected non critical ratio for other trisomics not involving the sex chromosomes, or ratios obtained when crossing  XX x XY diploids
 
 

f \ m

X

Y

X

XX

XY

1 male : 1 female

However, the sex chromosome transmission is low in pollen due to its length, allowing ratios deviating from the non critical 1:1 ratio. L5 was identified as the sex chromosome. X and Y look identical, in other words they are not heteromorphic.

Variety development

OP varieties: Historically, asparagus breeding has evolved from open pollinated (OP) varieties to different types of hybrid varieties with different degrees of sophistication.

Asparagus being a dioecious perennial, poses a real problem to chose the planting material which may be cropped for at least 10 years.

Initially, (50 years ago), OP varieties such as Mary Washington, were used. The problem was the heterogeneity of the variety, since seed was obtained from open pollinated plants in isolated plots without selection.

Clonal hybrids: With the advent of tissue culture techniques, F1 hybrids were produced by selecting good male and female parents that were mass propagated vegetatively and then crossed for F1 hybrid production. These are known as clonal F1 hybrids, since the parental plants are heterozygous for many loci, so some segregation still observed in the F1. Sometimes even F2 seed is also grown from these varieties as a cheaper alternative since F1 seed is expensive.

Most current commercial varieties are clonal F1 hybrids, which means that they consist of a heterogeneous mix of male and female plants.

To maintain F1 hybrid varieties, the parental plants must be maintained vegetatively or in vitro. Therefore, those producing these varieties must have tissue culture facilities.

Double hybrids: These were proposed as an attempt to avoid genetic heterogeneity in the variety and tissue culture steps.

Their development involves crossing a single male and a single female plant from say, varieties A and B, and selecting only male hybrid plants as parents for the double hybrid. The same is done from a different cross, say varieties C and D, for selection of females hybrids as parents for the double cross. These hybrids seems to have high yield potential and more uniformity than clonal hybrids. Another advantage of double hybrids is that they avoid problems of inbreeding depression which are observed when attempting to develop inbreds.

All-male varieties: Based on the rationale that in general male plants have higher yields than females in asparagus, a breeding scheme have been proposed to develop all-male asparagus varieties.

This is based in the construction of supermales or YY males, which will produce all-male hybrid XY progeny when crossed to XX females. Another advantage of these varieties is that the seed supplier has complete control over seed, because no seed can be produced by supermales.

Producing supermales: Supermale parents can be obtained by selfing andromonoecious plants (XY), which will segregate in a expected 1 XX: 2XY: 1YY ratio.

This is the most common approach. In order to distinguish XY from YY males, these are test crossed to XX females. XY will segregate 1male :1 female. YY will produce only male progeny.

Also andromonoecious XY can be used as females crossed to YY to generate 1:1 male andromonoecious. The problem is that the resulting male hybrids often show the andromonoeicous character because of initial selection for this trait. This will results in large berry set as in females, which translates into lower yields.

In-vitro culture techniques developed is asparagus opens up new alternatives to the breeder. These are anther culture and micropropagation that can be readily applied for hybrid production.

For example, supermales can be also obtained by anther culture of XY plants. Half of the doubled haploids are expected to be YY. Anther culture experiments by Favaligna where 131,925 anthers were cultured, a total of 880 YY plants were obtained (6.7 in 1000 anthers).

The main problem with anther culture is that not all plants respond well to this technique, which depends on plant genotype.

Once that supermales are obtained by either approach, these must be test crossed with various selected females for determining best combining ability.

Then all-male XY hybrids must be tested in the field.

The best YY parents must be mass propagated by in vitro technique to generate enough plants to produce enough hybrid seed.

Herbicides such as triazines, carbemates, alkanamidines and others can be used to induce early flowering for identifying supermales in shorter time. Sonoda et al.( 2003).

Tetraploid varieties: Polyploid breeding gaining interest in asparagus. For example, there is a tetraploid purple variety `Viola' which commands a higher price as an specialty crop. Schemes have been proposed to develop all-male 4x varieties, although these are complicated (see Skibe 1991).

Complexity of asparagus breeding:

Asparagus yields depend primarily on photosynthesis levels during the previous summer season. A high level of photosynthesis during the summer is essential for accumulation adequate food reserves in the storage roots. Additionally, warm temps (10 to 30C are necessary in the spring for fast spear growth. Asparagus has a strong apical dominance, which means that the next bud to grow on the bud cluster will not develop until the first shot has been harvested or developed in to a fern. Most European countries prefer white over green asparagus which are produced in high beds making production more expensive. Countries were spring temp is low use different strategies to move production to the summer from the spring. Ferns are allowed to grow until the middle of the summer, then cut to generate new spears. A variation of this is to let only a couple of spears develop into ferns, then harvest the next spears.

Breeding asparagus is a lengthily process since each test cycle

Each cycle takes at least five years:

1st year: Grow seedlings in nursery

2nd year: Establish crowns in the field following experimental design.

3rd year: Observations without harvesting.

4rd and 5th year: Yield evaluation.

Most US varieties derive from two var. developed by Norton in N.J. in 1906., Martha Washington and Mary W. which are resistant to rust. Therefore, they have a narrow germplasm base. Martha was more popular than Mary which never became very popular.

The more recent UC clonal hybrid varieties, such as the popular UC157 were developed from these early varieties.

Important traits and their evaluation:

Cylindrical spears,

Tight head

Oppressed scales

All green spear without anthocyanin

Smooth surface

Round shape spears

Selection in fern season is somewhat correlated with spears from next year. Plants with highest distance of branching in fern means tall spears, short distance, small spears.

Fern consist of modified leafs called cladophylls, which are the main photosynthetic organs.

Also diameter and number of stalks in fern season are correlated with number and diameter of spears next year.

Diseases:

Asparagus virus 1, aphid transmitted

Asparagus virus 2, seed transmitted.

Virus indexing is done with with Quenopodium plants.

Meristem culture is used for producing virus free plants

Fusarium is one of the main problems in asparagus and no source of resistance has been identified.

Most hybrids have field tolerance to fusarium by outgrowing the disease due to their higher vigor.

Phytophthora: no sources of resistance are known either for this disease.

Markers and mapping:

Markers have been developed fairly recently for asparagus. The first one were isozymes which allow variety identification. More recently, RFLPs, RAPDs and AFLPs have been developed and linkage maps have been constructed.

Phylogenetic relationships have been inferred by restricion fragment of chloroplast DNA, and by genome size and ITS rDNA (Stajner et al. 2002).

 

 

 

 

 

 


(based on Stajner et al. 2002)

Main applications:

Variety identification: These can be done using isozymes and DNA-based markers. Asparagus hybrid seed is very expensive and is a long term investment by the farmer. This is because the plants will be in the field for at least 10 years. Therefore, it is critical that the growers gets what they paid for. There have been instance for example where F2 seeds have been sold as F1 seed for UC157. Isozymes and RFLPs markers have been developed to distinguish F1 from F2 seed for this variety.

Gender markers: Markers for plant gender have been developed also in this crop. Therefore, it is now possible to select males and females at seedling stage. Further, YY supermales can be distinguished from XY males without the need of test-crossing. A total of 24 markers flanking the sex locus is now available. Some of these are co-dominant STS that can be disclosed by a simple PCR assay (Reamon and Jung, 2000). Jamsari et al 2004 derived sex determination markers from BAC clones. Nakayama et al (2006) derived a simple PCR assay from marker Asp1-T7 of Jamsari to distinguish males from females, however males could not be distinguished from supermales. Gebler et al (2007) reports a simple RAPD marker that could be useful to identify YY plants, but still needs to be confirmed. Telgmann et al (2007) produced genetic and physical map of the sex-determining locus with the goal to clone this gene. M locus is on the long arm of L5 in a low gene density area but rich on transposons. M region is ~2Mbp in size.

 

Transformation: Transformation has been accomplished by electroporation and particle bombardment to embryogenic callus (Cabrera Ponce et al. 1997).


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    Last modified:May 6, 2010

    Carlos F Quiros, 1998