Tropical Grasslands-Forrajes Tropicales (2017) Vol. 5(3):117–128 117

DOI: 10.17138/TGFT(5)117-128

Research Paper

Effect of pollination mode on progeny of Panicum coloratum var.

makarikariense: Implications for conservation and breeding

Efecto del modo de polinización sobre la progenie de Panicum coloratum

var. makarikariense : Implicaciones para conservación y fitomejoramiento

LORENA V. ARMANDO1,2, MARÍA A. TOMÁS1, ANTONIO F. GARAYALDE2,3 AND ALICIA D. CARRERA4

1 Instituto Nacional de Tecnología Agropecuaria (INTA), EEA Rafaela, Santa Fe, Argentina. www.inta.gob.ar

2 Centro de Recursos Naturales Renovables de la Zona Semiarida, CERZOS-CCT-CONICET, Bahía Blanca, Buenos Aires, Argentina. www.cerzos-conicet.gob.ar

3 Departamento de Matemática, Universidad Nacional del Sur, Bahía Blanca, Buenos Aires, Argentina.

www.matematica.uns.edu.ar

4 Departamento de Agronomía, Universidad Nacional del Sur, Bahía Blanca, Buenos Aires, Argentina.

www.uns.edu.ar/deptos/agronomia

Abstract

Panicum coloratum var. makarikariense, a perennial grass native to Africa, is adapted to a wide range of soil and climatic conditions with potential to be used as forage in tropical and semi-arid regions around the world. Our objective was to understand how the pollination mode affects viable seed production and further survival of the progeny. We evaluated self- and open-pollinated progenies from different accessions by measuring the seed production of the parents and their germination performance, germination rate and seedling survival. Parents and progeny were also fingerprinted with Simple Sequence Repeats (SSR). Progeny produced through open-pollination resulted in significantly more filled seeds and superior seedling survival than self-pollination. These results indicate that accessions studied here rely heavily on cross-pollination, whereas the contribution of self-pollinated offspring to the population is likely to be low. SSR profiles showed that, on average, 85% of the progeny (arising from cross-pollination) possessed paternal specific markers and 100% of them were genetically different from the maternal genotype. All plants examined had 4x = 36 chromosomes.

Overall, our findings indicate that var. makarikariense is able to generate highly polymorphic progeny through segregation and recombination. This study provides reference information for the formulation of appropriate strategies for pasture germplasm management, conservation and development of breeding programs.

Keywords : Breeding systems, pollination, genetic variation, germination, polyploidy, seed production.

Resumen

Panicum coloratum var. makarikariense es una gramínea perenne nativa de África. Se adapta a un amplio rango de ambientes y posee uso potencial como forraje en distintas regiones tropicales y semiáridas del mundo. El estudio tuvo como objetivo evaluar el efecto del modo de polinización sobre la producción de semilla viable y la supervivencia de la progenie. Se evaluaron progenies de autopolinización y de polinización cruzada en diferentes accesiones midiendo la producción de semillas, germinación, tasa de germinación y supervivencia de plántulas, y se obtuvieron perfiles moleculares con Secuencias Simples Repetidas (SSR). La progenie obtenida mediante polinización cruzada mostró ___________

Correspondence: L. Armando, Instituto Nacional de Tecnología

Agropecuaria, INTA-EEA, 2300 Rafaela, Santa Fe, Argentina.

E-mail: larmando@criba.edu.ar.

Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775)

118 L.V. Armando, M.A. Tomás, A.F. Garayalde and A.D. Carrera

significativamente mayor producción de semillas llenas y supervivencia de plántulas que la de autopolinización. Esto indica que las accesiones evaluadas dependen en gran medida de la alogamia y que la contribución de la descendencia por autofertilización a la población sería escasa. Los perfiles moleculares SSR mostraron que, en promedio, 85% de la progenie (obtenida a partir de polinización cruzada) presentó marcadores específicos paternos y 100% de ella difirió del genotipo materno. Todas las plantas examinadas presentaron 4x = 36 cromosomas. En conjunto, los resultados indican que la var. makarikariense puede generar progenie altamente polimórfica a través de la segregación y recombinación.

Este estudio provee información útil para el diseño de estrategias de conservación, manejo del germoplasma y programas de mejoramiento.

Palabras clave : Germinación, polinización, poliploidía, producción de semilla, sistema de reproducción, variación genética.

Introduction

their evolutionary potential. This information is critical

when planning and developing conservation and breeding

The amount of genetic variability within a species and,

programs.

therefore, adaptability of their progeny to the environ-

Panicum coloratum L., a perennial grass native to

ment, are mostly determined by the breeding system.

Africa, is adapted to a wide range of soil and climatic

Autogamous and asexual species produce populations

conditions, and has been used as forage in Australia,

with little evolutionary flexibility and high local

Japan, USA, Mexico and South America (Cook et al.

specialization (Stebbins 1950), whereas outcrossing

2005). This species has been classified into mainly 2

species produce more genetically diverse and ecologically

botanical varieties, var. makarikariense Gooss. and var.

variable offspring. Grasses display an extraordinary

coloratum, distinguished by morphological traits and

diversity of breeding systems including outcrossing,

environmental preferences (Bogdan 1977; Armando et al.

selfing or mixed-breeding, and a mixture of asexual and

2013). The var. makarikariense is particularly well

sexual reproduction (Quinn 1998). Many plant species

adapted to heavy clay soils that fluctuate between drought

have developed different ecological, morphological and

and waterlogged conditions, whereas var. coloratum

physiological mechanisms that reduce the degree of self-

develops well in sandy soils, is tolerant of salinity and

fertilization to promote cross-pollination (Eckert 1994),

performs well at higher latitudes or elevations, as it

most likely motivated by the increase in individual and

thrives under low temperatures, withstanding some frost

(Tischler and Ocumpaugh 2004). In Argentina, a breeding

average population fitness caused by heterosis.

program

and

research

activities involving

var.

The frequency of outcrossing is an important deter-

makarikariense were initiated by the National Institute of

minant of population genetic structure, affecting both

Agricultural Technology (INTA) in 2006, with the

genetic diversity within populations and genetic

purpose of developing new pasture cultivars adapted to

differentiation among them (Barrett and Harder 1996).

marginal (drought, waterlogging, salinity or thermal

Methods commonly employed for assessing the mode of

stress) and less productive environments where livestock

reproduction in forage grasses include cytological and

production has been displaced, with expansion of

embryological analyses of the mother plant and screening

cropping into the most productive paddocks and planting

for morphologically aberrant progeny. Molecular marker

of soybeans.

analysis, in particular, Simple Sequence Repeat (SSR) or

Panicum coloratum botanical varieties have been

microsatellite, is a tool now widely used in a variety of

described as mainly allogamous (Brown and Emery 1958;

fundamental and applied fields of biology, including the

Hutchison and Bashaw 1964), although the degree of self-

identification of selfed, outcrossed or apomictic progeny

fertilization has not been quantified and apomictic

in several grass species (Chistiakov et al. 2006; Liu and

mechanisms have been suggested (Hutchison and Bashaw

Wu 2012). SSRs are loci ubiquitously distributed within

1964). Unlike previous reports, which focused on the

genomes that show a high level of polymorphism,

female parts of flowers and embryo sac development, our

environmental independence and rapid detection pro-

main interest is the analysis of the particular effects of

tocols. The reproductive system and the ploidy level of a

different pollination systems on viable seed production

species determine the transmission of genes across

and the survival of subsequent progeny. In addition,

generations, the pattern of inheritance and gene flow, and

cytogenetic studies in var. makarikariense showed

influence the genetic structure of plant populations and

variable numbers of chromosomes: 2n = 18, 36, 45, 49

Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775)

Pollination mode and progeny of Panicum coloratum 119

and 63 (Hutchison and Bashaw 1964; Pritchard and De

sion were placed at 0.6 m intervals in an 8 × 4 matrix plot,

Lacy 1974).

with plots 15 m apart, while the 15 IFF genotypes were

In the present work, progeny of P. coloratum var.

clonally propagated 8 times and arranged linearly in an

makarikariense derived from self- and open-pollinated

8 × 15 matrix plot at a distance of 0.6 m.

panicles were studied through the stages of seed

production, germination and progeny survival. Addi-

Seed production

tional data were obtained from SSR marker analysis,

and chromosome number was also determined. This

Seed production of 3 plants (only 2 plants for accession

study attempted to provide information regarding

DF), selected at random from each of the UCB, MR, BR,

the reproductive behavior of P. coloratum var.

ER and CM accessions and 1 clone of each of the 15 IFF

makarikariense, with utility for conservation and

genotypes (Table 1) (a total of 32 plants) of P. coloratum

breeding.

var. makarikariense, was measured in the field from

March to May 2009. Unfortunately, 1 plant of the DF

Materials and Methods

accession was damaged and data were unavailable. For

each plant, 2 panicles were selected at random: 1 for self-

Plant samples

pollination and 1 for open-pollination. Only a single

panicle was enclosed in each seed trap, the ones for self-

Panicum coloratum was introduced into Argentina in the

pollination before anthesis and the ones for outcrossing

1990s but has not been used widely as forage, although it

when 2/3 of the panicle was in anthesis. Seed traps were

has been conserved at various locations as collections or

used in order to facilitate seed collection and to prevent

in small paddocks. Details of introductions are often

losses by seed shattering. Traps were therefore put in

limited, with many coming from different parts of the

place at different stages of development for self- and

world. A collection of P. coloratum var. makarikariense

open-pollinated treatments, but within the same treat-

(Table 1) was established in a common garden at the

ment attempts were made to select panicles at the same

INTA Rafaela Experiment Station (31°11'41'' S,

stage of development. In self-pollinated treatments seed

61°29'55'' W) in Argentina in 2006, as a breeding popu-

traps were covered with a white cotton bag to prevent

lation. Pre-breeding studies demonstrated a high level of

pollen arrival from other sources without precluding light

variability in morphological and molecular markers, both

interception and photosynthesis of glumes (Figure 1).

among and within accessions, which justified the

Self- and open-pollinated seeds were collected simulta-

initiation of a breeding program (Armando et al. 2013). In

neously once a week and manually separated from the

fact, a cultivar from the program was released recently:

glumes and other residuals. Eventually, the total number

Kapivera INTA (Giordano et al. 2013). The collection

of seeds per inflorescence was counted, i.e. dark brown

comprised 6 accessions of 32 plants each and 15 clonally

seeds (comprising lemma and palea containing a

propagated genotypes (IFF) obtained by selection on

caryopsis). Small light-weight whitish seeds (hereafter

agronomic characteristics. The 32 plants of each acces-

referred to as “empty seeds”) were also produced and

Table 1. Accessions of P. coloratum var. makarikariense and their collection site description.

Accession code

Description

Site of preservation

Coordinates

Province

DF

Twelve-year-old pasture

Dean Funes (150 km Northwest

30°26' S, 64°21' W

Córdoba

Under heavy cattle grazing

from Córdoba city)

UCB

Ungrazed pasture

Catholic University of Córdoba;

31°25' S, 64°11' W

Córdoba

collected in South Africa

MR

Ungrazed pasture

Catholic University of Córdoba;

31°25' S, 64°11' W

Córdoba

collected in South Africa

BR

Ten-year-old pasture under cattle

Mercedes Experiment Station

29°11′ S, 58°02′ W

Corrientes

grazing

(INTA); introduced from Brazil

ER

Five-year-old pasture under cattle

Private farm near Mercedes

29°03' S, 57°49' W

Corrientes

grazing

IFF 1‒15

Clonal materials

CIAP-INTA Institute of Physiology

31°24' S, 61°11' W

Córdoba

and Plant Genetic Resources

CM

Seeds commercially distributed by a cv. ‘Bambatsi’; imported from

-

private company

Australia

Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775)

120 L.V. Armando, M.A. Tomás, A.F. Garayalde and A.D. Carrera

counted as immature florets and/or spikelets with pre-

the initiation of the germination trial. Studies by Tomás et

mature shattering from the inflorescences. Empty seeds

al. (2015) showed that maximum germination has been

show poor germination capacity, while dark brown seeds

reached by day 7. A seed was considered germinated

show a high germination percentage (Maina et al. 2017).

when the radicle emerged through the seed coat. Eight-

The final numbers of seeds produced under self- and

day-old seedlings were individually transplanted into 0.5

open-pollinated conditions were compared.

L plastic containers filled with a soil-sand-perlite mix

(1:1:1 v/v), placed in a greenhouse at 28 °C and watered

as needed, usually every 2 to 3 days. Seedling survival

percentage (% Ss) was recorded when seedlings were 15

and 40 days old.

Progeny test

In order to analyze genetic composition of the offspring,

a random sample of 12‒15 seedling descendants from 3

female parents, UCB3, ER1 and IFF10, was genetically

characterized. These plants were selected to represent

the observed range in the number of seeds produced

within var. makarikariense (see Figure 2). Progeny test

was performed only on seeds produced via open

pollination as only a limited number of progeny were

Figure 1. Seed traps enclosing inflorescences consisting of an

obtained from selfing. In addition, progeny obtained from

iron cylindrical structure covered by a nylon stocking (modified

open-pollinated traps resembled more natural pollination

from Young 1986). a) Open-pollination trap (left) and self-

conditions.

pollination trap with a white cloth bag (right). b) Detail of the

DNA extraction was carried out using a modified SDS

lower part of the trap (water drainage). Seeds (= mature florets)

method (Edwards et al. 1991). Approximately 150 mg of

were trapped and funneled into a cap as they shattered from the

leaf tissue (from plants >1 year old) was homogenized in

panicle.

liquid nitrogen. A 700 μL volume of extraction buffer

containing: 50 mM Tris pH 8, 10 mM EDTA pH 8,

Seed germination and seedling survival

100 mM NaCl, 10 mM 𝛽-mercaptoethanol and 10% SDS,

was added and incubated at 65 °C for 20 min. After

Harvested seeds were naturally air-dried and stored at

adding 200 μL of 5 M potassium acetate pH 4.8, the

room temperature in paper bags for 1 year before testing

sample was incubated on ice for at least 20 min and then

for seed germination to ensure dormancy was already

centrifuged at 13,000 rpm for 20 min. This was followed

overcome (Tischler and Young 1987). Of the 32 plants

by precipitation with 700 μL of iso-propanol incubated

evaluated, only 11 produced filled seeds under self-

at -20 °C for 10 min, and centrifugation at 13,000 rpm for

pollination. In each accession, only plants producing a

4 min. The resulting pellet was washed with ethanol 70%

good quantity of filled seeds (UCB3, MR1, BR1, ER1,

and dissolved in 100 μL of 1 x TE buffer. DNA quality

CM2, IFF10; see Figure 2) were used to evaluate

germination and seedling survival (n = 6). Thirty filled

was evaluated in agarose gel and the quantity was

seeds per panicle from the same plants in both self- and

determined by spectrophotometry.

open-pollinated treatments were placed in 10-cm

In previous work, out of 40 heterologous SSR loci

diameter Petri dishes separately with filter paper at the

evaluated in P. coloratum var. makarikariense, 10 primer

bottom moistened with distilled water, and incubated in a

pairs were successfully amplified showing polymorphic

programmed germination chamber at 42% humidity and

and clear banding patterns (Armando et al. 2015). From

27 °C (Tomás et al. 2015) at a 16-hour photoperiod (light

these, the 5 most variable ones were chosen for analysis

photon flux density: 48 mmol/s/m2). Dishes from

both of mother plants and offspring (Table 2).

different pollination treatments and different plants were

Amplification reactions were performed in 20 μL final

randomly arranged in the chamber. The number of

volume containing: 30 ng of DNA template, 2.5 mM

germinated seeds per dish was counted daily and seed

MgCl2, 0.125 mM of each dNTPs, 10 pmol of each primer

germination percentage (% G) was recorded on day 7 after

and 1 U of Taq DNA polymerase in 1.6x buffer. Negative

Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775)

Pollination mode and progeny of Panicum coloratum 121

Table 2. Simple sequence repeat (SSR) loci used for progeny analysis and polymerase chain reactions (PCR) conditions.

Repeat motif

Source

Sequence (5´  3´)

TD/Tm

1- (AG)8T(AG)7

EST- Panicum maximum

F: TGTATGAGCTGAGTCGC

63–53/58

R: TGGTAATCTAGTTGATATTC

2- (AG)8

EST- Panicum maximum

F: CCCGAGGCGATCCGATTCGTT

63–53/58

R: TACGCCGACGACGAGGACGA

3- (AT)13

EST- Panicum virgatum

F: TCCAGATGACTCCCAGGAAC

50–40/45

R: TCATCACTCGATTCCTCAAGC

4- (GT)38

Genomic- Panicum virgatum

F: GCAACCATGACAAGAAGCAT

63–53/58

R: ATACAAACCGGGGTGCTAAG

5- (CGT)n

EST- Eragrostis curvula

F: TCTCCAACACGCCACGAC

63–53/58

R: CAATCCACTACAAGAAACCAC

SSR 1 and 2 (Ebina et al. 2007); 3 (Tobias et al. 2006); 4 (Wang et al. 2011); 5 (Cervigni et al. 2008).

TD/Tm: Touchdown/Annealing temperature; F: forward primer; R: reverse primer.

controls (no DNA template) were also included. Poly-

removed from the roots, placed in a drop of 2% hema-

merase chain reactions (PCR) were carried out in an MJ

toxylin with 2% ferric citrate used as mordant, and

Research Thermal Cycler. The optimum annealing

squashed (modified from Núñez 1968). Cells with fully

temperature (Tm) was determined for each locus (Table

contracted and well spread metaphase chromosomes were

2). By touchdown PCR (TD), the annealing temperature

photographed using a digital camera.

was decreased to 1 °C starting with 5 °C over the set

annealing temperature. Initial denaturation step of 95 °C

Data analyses

for 3 min was followed by 10 touchdown cycles of 94 °C

for 30 sec, touchdown annealing temperature for 30 sec

Average number of filled and empty seeds, germination

and 72 °C for 45 sec. PCR products were subsequently

percentages at day 7, time at which 50% of seeds had

amplified for 34 cycles at 94 °C for 30 sec, annealing

germinated (T50) and survival of 15- and 40-day-old

temperature for 30 sec, and 72 °C for 45 sec with a final

seedlings were recorded for self- and open-pollinated

extension at 72 °C for 20 min. Amplifications were

conditions. The mean values obtained for the 2 forms of

initially checked on 1% agarose gels. PCR products were

pollination were compared through one-tailed Student’s t-

analyzed on 6% denaturing polyacrylamide gel, with a

test for paired samples, since a higher number of filled

TBE 1x electrophoresis buffer at 50W for 1 h and 45 min

seeds was expected in cross-pollination. Analysis of

to 2 h. Bands were visualized by silver staining and

variance (ANOVA) for the mean number of seeds was

scanned.

performed and accessions were compared by Fisher’s

LSD tests. Statistical analyses were performed using

Chromosome number

Infostat software (Di Rienzo et al. 2008).

Based on the number of seeds, germination and

To confirm the chromosome number of P. coloratum var.

survival data per panicle, probability of seed production,

makarikariense, 8 different plants were evaluated. One plant

and probability of seedling survival for self- or cross-

from each accession was chosen at random and considered

pollinated conditions were estimated by using the

representative of each accession (DF, UCB, MR, BR, ER

and CM), while 2 plants were selected from IFF.

conditional probability and Bayes Theorem (Quinn and

Root tips of 3-month-old plants were pretreated with

Keough 2002). The probability of seedling survival (SS)

8-hydroxyquinoline (0.002 ml/g) for 5 h. The roots were

was calculated as: P(SS) = P(SS/SPs)*P(SPs) +

fixed in a freshly prepared mixture of ethanol:glacial

P(SS/SPo)*P(SPo), where P(SS/SPS) and P(SS/SPO) are

acetic acid (3:1 v/v) for 48 h at room temperature and then

the conditional probabilities of seedling survival from

placed in 70% ethanol at 4 °C for several weeks. Treated

seeds produced under self- and open-pollinated condi-

roots were put in 95, 70 and 40% ethanol and distilled

tions, respectively; P(SPs) and P(SPo) are the probabil-

water for 15 min each, hydrolyzed in 1 N HC1 at 60 °C

ities of seed production under self- and open-pollination;

for 8 min, transferred to distilled water for 2 min and

and P(SS) is the probability of seedling survival. The

stained in leuco-basic fuchsin for 1 h at room temperature

conditional probability of finding a plant given that it was

in the dark. The 3–4 mm deeply stained root tips were

produced under self- or open-pollination was calculated

Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775)

122 L.V. Armando, M.A. Tomás, A.F. Garayalde and A.D. Carrera

as: P(SPs/SS) = P(SS/SPs)*P(SPs)/P(SS) and P(SPo/SS)

per panicle was highly variable among plants, registering

= P(SS/SPo)*P(SPo)/P(SS), respectively.

values from 0 to 72 (CV = 149%) for self-pollinated

In the progeny test, offspring’s DNA fingerprints were

panicles and from 14 to 795 (CV = 61%) for open-

individually compared with the maternal pattern. The

pollinated panicles (Figure 2). Mean number of filled

occurrence of a sexual reproduction event was defined as

seeds differed significantly among accessions for both

when progeny DNA fingerprints revealed a deviation

self- and open-pollination methods (Figure 3). According

from the maternal profile. Each band was considered as

to Fisherś LSD test, the commercial variety (cv.

an independent locus, and polymorphic bands were

Bambatsi) had the highest mean number of filled seeds

scored visually as either absent (0) or present (1) for each

under open-pollination, while the lowest number was

of the 45 plants. Only those bands consistently scored

from accession BR (Figure 3).

were considered for analysis. By combining the markers,

Regarding progeny performance, the seed germination

unique profiles were obtained for each individual. Genetic

percentages were above 80% and together with T50 no

diversity in the progeny was estimated using the total

significant differences were observed in filled seeds

number of alleles, number of alleles shared with female

obtained from both self- and open-pollinated conditions

parents (maternal alleles) or deriving from male parents

(Table 3). In all the plants evaluated T50 values showed

(paternal alleles), and percentage of polymorphic loci

that at least half of the seeds (range 50–97%) had

(%P).

germinated by the 3rd day after the trial started, except for

The genetic dissimilarity between the maternal parents

one plant where T50 extended to the 5th day (data not

and their progeny was analyzed using a genetic binary

shown). The survival of both self- and open-pollinated

distance (GD) according to Huff et al. (1993). An

seedlings decreased over time, but the survival of seedlings

UPGMA cluster was obtained from GD matrix. Analyses

derived from open-pollination was significantly higher

of molecular data were performed using GenAlEx 6,

(P<0.001) than those obtained from selfing at both 15 and

Genetic Analysis in Excel (Peakall and Smouse 2012) and

40 days of age. In particular, survival of 40-day-old

Infostat programs (Di Rienzo et al. 2008).

seedlings from self-pollinated panicles was much lower

than that from out-crossing (4.8 vs. 35.7%) (Table 3).

Results

Estimates of total seed production and seedling survival

of plants from accessions of var. makarikariense are shown

Effect of pollination method on progeny number and

in Table 4, differentiating those obtained from the different

seedling survival

methods of pollination. Of the total number of filled seeds

produced per panicle, the probability of them being

The mean number of filled seeds per panicle under open-

produced by open-pollination was greater (92%) than by

pollination was considerably higher than those produced

self-pollination (8%). The estimated survival at 40 days of

under self-pollination (P<0.001, Table 3). In contrast, the

a seedling from a seed obtained by self-pollination was

mean number of empty seeds (empty perfect florets

lower (6%) than the survival of a seedling coming from a

and/or caryopses unable to germinate) did not differ

seed obtained by open-pollination (32%). In addition, the

significantly between the 2 forms of pollination (P =

estimated probability of plant survival for this period of

0.1518, Table 3). In general, the number of filled seeds

time, independently of whether it came from self- or open-

Table 3. Comparison of the average number of filled (n° Fs) and empty (n° Es) seeds per panicle, seed germination percentage (% G), time needed for seeds to reach 50% germination (T50) and seedling survival percentage (% SS) of 15- and 40-day-old seedlings, between self-pollinated and open-pollinated panicles, using the student t-test in P. coloratum var. makarikariense.

n° Fs

n° Es

% G

T50

% SS 15

% SS 40

n

32

32

6

6

6

6

Mean Self

10.9

176.1

88.5

80.1

38.1

4.78

Mean Open

279.2

207.6

81.7

85.6

77.6

35.73

Mean difference

-268.3

-31.5

6.83

-5.48

-39. 5

-31.0

T

-9.13

-1.05

0.91

-1.94

-10.15

-5.80

P

<0.0001

0.1518

0.7980

0.0551

0.0001

0.0011

n: number of genotypes evaluated.

T: Student t-values. P: p value of student t-test.

Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775)

Pollination mode and progeny of Panicum coloratum 123

Figure 2. Number of filled seeds produced per panicle under self-pollination and open-pollination in 32 individuals of P. coloratum var. makarikariense.

Figure 3. Mean number of filled seeds produced per panicle under self-pollination and open-pollination in accessions of P. coloratum var. makarikariense. In each type of pollination, different letters indicate significant differences between accessions (P<0.001) using Fisher’s LSD tests and bars represent standard errors of means (lower case letters refer to open-pollinated and upper case to self-pollinated).

Table 4. Probabilities of seed production (SP) under self- (S) or open-pollination (O) and seedling survival (SS) in P. coloratum var.

makarikariense.

Pollination

Probabilities

UCB

MR

BR

ER

CM

IFF

Mean

Self

p(SPS)

0.29

0.09

0.09

0.11

0.03

0.05

0.08

p(SS/SPS)

0.13

0.00

0.00

0.03

0.00

0.10

0.06

p(SPS/SS)

0.15

0.00

0.00

0.01

0.00

0.02

0.03

Open

p(SPO)

0.71

0.91

0.91

0.89

0.97

0.95

0.92

p(SS/SPO)

0.30

0.23

0.36

0.53

0.17

0.37

0.32

p(SPO/SS)

0.85

1.00

1.00

0.99

1.00

0.98

0.97

-

p(SS)

0.25

0.21

0.32

0.48

0.16

0.35

0.30

p(SS/SPS) and p(SS/SPO): conditional probability of seedling survival given that seed production was under self- or open-pollination, respectively.

p(SPs/SS) and p(SPO/SS): conditional probability of finding a plant given that it was produced under self- or open-pollination, respectively.

Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775)

124 L.V. Armando, M.A. Tomás, A.F. Garayalde and A.D. Carrera

Figure 4. Percentage of SSR alleles attributed to female ( ) and male ( ) parents in progenies of P. coloratum var. makarikariense from 3 accessions (names are detailed in Table 1).

pollination, was 30%. Combining data, the estimated prob-

maternal and paternal alleles could be recognized in

ability of finding a plant obtained by open-pollination was

individual progeny (Table 5). On average, 85% (range

significantly higher (97%) than those produced by self-

67–100%) of individual progeny presented alleles not

pollination (3%).

found in the female parent (Figure 4). Additionally,

offspring were not identical with their maternal patterns

Effects of pollination method on genetic variability and

but they were closely grouped in the dendrogram

contrast with female parents

according to families (Figure 5). Moreover, the 3 female

plants were genetically distinct.

Progeny from 3 female parents were analyzed with 5 SSR

markers. Offspring plants were classified as identical

Chromosome numbers in the P. coloratum var.

when patterns were the same as the female parents at all

makarikariense collection

the evaluated SSR loci or distinct when any band

differences between female parent and progeny were

In all mitotic cells observed, 36 chromosomes were

observed. The SSR analysis showed values of percent

counted at metaphase. This number remained stable for

polymorphic loci (%P) over 50% in progenies, and

all 8 P. coloratum var. makarikariense plants evaluated.

Table 5. Genetic diversity in 3 progeny of P. coloratum var. makarikariense assessed by 5 simple sequence repeat markers (SSR).

Female parent

N° progeny

Total alleles

Maternal alleles

Paternal alleles

%P

UCB3

15

22

14

8

50.0

ER1

15

23

12

11

56.3

IF10

12

25

16

9

62.5

%P: percentage of polymorphic SSR loci in progeny.

Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775)

Pollination mode and progeny of Panicum coloratum 125

Figure 5. Unweighted pair-group method with arithmetical average (UPGMA) dendrogram based on the GD SSR matrix of 3

female parents (F) and their progeny (P) in families of P. coloratum var. makarikariense. The female plants are underlined.

Discussion

production per panicle among genotypes from different

accessions in the collection (Barrios et al. 2010). These

There is an increasing need for a better understanding of

accessions are also distinctive in other morphological

the reproductive biology in P. coloratum, given that this

characters, both vegetative and reproductive (Giordano et

is an extremely important prerequisite to develop breed-

al. 2013; Armando et al. 2013; 2015). In addition, seed

ing strategies and also for germplasm management and

yield is a complex character and additional variability

conservation purposes. In this study, we sought to

arises as seed set is the culmination of a series of process-

determine the preponderant mode of reproduction of one

es at canopy level including radiation interception, bio-

collection of the var. makarikariense used for breeding

mass production and partitioning. Therefore, variability

purposes both by evaluating seed production in open and

among individuals may be the result of both genetic

forced-to-inbreed panicles and by a progeny test using

variation and the integration of these different genetic

SSR markers. The evaluated plants in this study were

backgrounds with the multiple environmental influences

selected to cover the variability present in the germplasm

(Boelt and Studer 2010). The considerable differences in

collection at INTA EEA Rafaela, which comprises

seed production we observed among plants and

accessions collected within semi-arid temperate mesic

accessions suggest a promising scenario for selection to

and subtropical zones, both grazed and non-grazed areas,

increase seed production by means of augmenting the

and a commercial variety. Although we analyzed only 3

number of seeds per panicle.

panicles per accession, our results corroborate previously

The results from seed production, germination and

reported studies pointing out extensive variability in seed

seedling survival assessments in var. makarikariense

Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775)

126 L.V. Armando, M.A. Tomás, A.F. Garayalde and A.D. Carrera

indicated that open-pollination is by far the most frequent

Progeny without clear paternal contribution were

form of pollination in the reproductive biology of this

probably derived from self-pollination or open-

variety. A similar behavior is suggested for var.

pollination involving parents with the same molecular

coloratum based on a small sample of 3 plants (data not

pattern, although this value may be reduced with an

shown). Additionally, as only a few panicles produced

increased number of markers. In addition, the level of

good quality seeds by self-pollination, a limited number

genomic DNA polymorphism of the analyzed progeny

of plants could be analyzed. However, the results from the

suggests sexuality and genetic recombination, and

combined probabilities clearly showed that progeny from

provides additional evidence that P. coloratum is mainly

selfing make low to no contribution to the population over

an allogamous species.

time since a mean of only 6% of the plants obtained from

All our analyses accumulated evidence pointing to

self-pollinated seeds survived. Although seeds obtained

P. coloratum var. makarikariense as a species with

under self-pollination were not weighed, they were

mainly sexual reproduction that depends primarily on

visibly much smaller than and had a different color (white

open-pollination to obtain viable offspring. All var.

vs. brown) from those produced by open-pollination,

makarikariense plants evaluated in this study had 36

which could explain the lower vigor of the seedlings

chromosomes, and the same number was observed in 3

(Tomás et al. 2007). The fact that we did obtain seeds

plants of var. coloratum (data not shown). Considering a

from selfing demonstrates the existence of some degree

basic number of x = 9 (Hamoud et al. 1994), these plants

of self-compatibility. Burson and Young (1983), using

are tetraploids. This ploidy level is one of the most

fluorescence microscopy, demonstrated that, when var.

frequently reported for P. coloratum (Hutchison and

coloratum was self-pollinated, 90% of the pollen germi-

Bashaw 1964; Pritchard and De Lacy 1974). Apomixis

nated within minutes after the pollen grain came in

occurs throughout the plant kingdom and is always

contact with the stigma, but only 2% of the pollen tubes

associated with polyploidy as was reported in Panicum

actually grew into the ovary and entered the micropyle

maximum and Paspalum notatum (Warmke 1954; Quarin

within 1 hour after pollination, suggesting active self-

et al. 2001). However, sexuality also occurs at the 4x and

incompatibility mechanisms. A gametophytic S-Z in-

6x ploidy levels as in Panicum virgatum and Brachiaria

compatibility system has been found in related species

humidicola (Barnett and Carver 1967; Pagliarini et al.

(Martinez-Reyna and Vogel 2002) and is common for

2012). The natural distribution of diploid and tetraploid

most of Poaceae (Baumann et al. 2000). In the majority of

levels in P. coloratum at its center of origin appears to

the grasses this system is not absolute, and some seeds

occur throughout central and South Africa, while the

may be obtained from self-fertilization. Further, selfed

hexaploid level was confined to East Africa (Pritchard

progeny of highly heterozygous genotypes could result in

and De Lacy 1974).

fitness reduction due to inbreeding (Eckert 1994), a point

Knowledge of the chromosome number and ploidy

that we cannot prove with only one generation of selfing.

level of the germplasm is necessary for developing an

Brown and Emery (1958) observed typical sexual 8-

efficient strategy of preservation of this promising forage

nucleate embryo sacs in 82 ovules of var. makarikariense

species and is crucial for use in the P. coloratum breeding

and var. coloratum plants indicating both varieties

program. The ploidy level of this species may partially

reproduced sexually . Hutchison and Bashaw (1964) also

explain the high level of genetic variability in the species

observed 8-nucleate embryo sacs in both varieties but

and the highly polymorphic progeny. New genetic

about 2% of the older ovules had large vacuolated cells,

combinations seem to be obtained relatively easily

which appeared similar to multiple embryo sacs. They

through segregation and recombination. This fact may

considered this as possible evidence of apospory, but

represent an interesting means to increase germplasm

developed embryos were not observed in these cells,

variability and may be an important factor to consider

which dispelled the possibility of apomixis in this species.

when releasing new materials resulting from selection and

The high amount of variation expressed in open-

breeding. In addition, the mode of reproduction in a given

pollinated makarikariense progeny rules out the

species must be clear to the plant breeder to accomplish

possibility of apomictic reproduction in the species. In our

crop improvement. Knowledge about how a plant

study, molecular progeny tests revealed that an average of

reproduces naturally helps the breeder to predict the

85% of offspring possessed paternal alleles; this is

behavior under field conditions and to establish the most

indicative of cross-pollination and 100% of them were

appropriate selection method, which markedly differs

genetically distinguishable from the maternal genotype.

between self- and cross-pollinated crops. Knowledge of

Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775)

Pollination mode and progeny of Panicum coloratum 127

breeding systems is also of benefit for germplasm banks.

Bogdan AV. 1977. Tropical pasture and fodder plants (grasses

Based on our results, the number of individuals destined

and legumes). Tropical Agriculture Series, Longman Group,

for seed increase should be large enough to include the

London, UK.

Brown WV; Emery WHP. 1958. Apomixis in the Gramineae:

original variability in genetic diversity. Moreover,

Panicoideae. American Journal of Botany 45:253–263.

isolation distances need to be taken into account in order

www.jstor.org/stable/2439258

to prevent gene flow among accessions and thus preserve

Burson BL; Young BA. 1983. Pollen-pistil interactions and

the genetic identity of each accession.

interspecific-incompatibility among Panicum antidotale, P.

coloratum, and P. deustum. Euphytica 32:397–405. DOI:

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(Received for publication 30 December 2016; accepted 16 August 2017; published 30 September 2017)

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Tropical Grasslands-Forrajes Tropicales (ISSN: 2346-3775)