Germplasm exploitation for citrus genetic improvement

Marco Caruso

CREA - Council for Agricultural Research and Economics

marco.caruso@crea.gov.it

Research Centre for Olive, Citrus and Tree Fruit, Acireale, ITALY

CREA-ENEA-Israel Workshop, Rome 7-8 March 2019

Citrus breeding at CREA: cultivar release

Pigmented oranges Mandarins (clementine selections, diploid

and triploid hybrids) Lemons Rootstocks Sanitation and evaluation of local and

foreign selections

Started in the 1940’s. At least three generations of breeders involved. Many varieties released to the growers

Citrus (625) Citrus relatives (47)

Sweet orange (265)

Bergamot (3)

Lemon (103)

Lemon-like (9)

Kumquat (5) Poncirus & hybrid (33) Eremocitrus

(1) Microcitrus (4) Murraya (1) Severinia (1) Citropsis (2)

Chinotto (2)

Sour orange (28)

Clementine (38)

Mandarin (66)

Grapefruit (27)

Pummelo (8) Tangelo (10)

Tangor (4) Satsuma (4)

Lime/limetta (16) Citron (6)

Calamondin (2)

Other citrus (43)

Lemon (94)

Moro (15) Sanguigno, sanguinello (35)

Tarocco (108)

Blood (156)

Other sweet oranges (109)

Valencia (11) Ovale (8) Vaniglia (5) Other common (15) Navel (50)

The CREA citrus germplasm

Sicily has been a centre for clonal diversification for blood oranges and lemons due to the long history of cultivation

Despite a wide range of differences in horticultural traits, all (in case of sweet orange) or almost all (in case of lemon) variability was determined by somatic mutations

From germplasm conservation to its utilization

evaluation of fruit quality and

year-to-year stability of pulp pigmentation

Whole genome resequencing for clonal fingerprinting and identification of causative mutations for pomological traits

Looking for mal secco tolerance

or resistance Generation of segregating

populations for mal secco suceptibility (resistant species × susceptible lemons)

The blood orange germplasm

The lemon germplasm

Blood orange germplasm consist of several clones differing for ripening period, and internal and external coloration, however:

Pigmentation is cold dependent

Exposure, blooming period, position of the fruits in the canopy, cultural practices and rootstock represent additional variability elements

Clonal selections, and their interaction with the environment

The exhaustive phenotypic characterization is

hampered by such influence of external factors

Environment influences fruit color

Sun red mandarin, Florida, 12 dec 2018

VS

Sun red mandarin, Sicily, 20 nov 2018

Color is influenced by the rootstock

Carpenter

Furr

F6P12

Swingle

Bitters (C22)

C. macrophylla

Mandared gratfed onto different rootstocks

Blood orange germplasm at CREA

~110 different clones (nucellar lines

under evaluation and gamma-ray

irradiated plants are excluded)

Most grafted in 2010 on Swingle

citrumelo planted in 2006. New

clones added yearly

Consisting of old lines, sometimes

chimeras are evident; some

nucellar selections are also

present.

Block 2b (germplasm):

> 90 genotypes

Vaccaro

Tarocco a buccia gialla

Stef. Fontanazza

Variegato

Guzzardi

Sanguinello

comune

Sanguinello da

Spina II

We estabilished a germplasm

collection that allows a comparison

among several blood orange clones

(same age, same rootstock, same

block, same cultural practices).

Block7 : 20 genotypes

Tarocco varieties (planted in 2003, grafted on

Carrizo citrange)

Virus free material obtained by micro-grafting

and nucellar selection

Blood orange germplasm at CREA

Tarocco Rosso Tarocco TDV Tarocco Sciara Tarocco Meli

Mean Standard deviation Standard error Range min Range max

Fruit weight (g) 189.24 31.01 3.27 109.61 271.99

equatorial diameter (mm) 7.21 0.41 0.04 6.10 8.42

polar diameter (mm) 7.29 0.58 0.06 6.18 8.60

seed no. 1.07 1.32 0.14 0 5

juice (%) 47.78 5.89 0.62 30.05 68.29

TSS (° Brix) 11.54 0.84 0.09 9.50 13.83

pH 3.41 0.28 0.03 3.10 5.76

Acidity (%) 1.19 0.21 0.02 0.10 1.67

Anthocyanin (mg l-1) 20.04 19.64 2.07 0.00 75.62

L* peel 60.43 4.54 0.48 45.60 71.20

a* peel 35.35 3.68 0.39 21.77 39.79

b* peel 57.84 6.37 0.67 35.12 71.57

Peel colour index 10.68 2.99 0.32 4.27 22.90

L* pulp 37.50 5.92 0.62 25.03 58.37

a* pulp 15.62 3.33 0.35 7.46 22.88

b* pulp 31.27 8.02 0.85 9.99 49.93

Pulp colour index 18.58 13.84 1.46 3.39 84.98

width of the central axis (mm) 9.64 1.85 0.20 2.87 14.15

peel thickness (mm) 4.89 0.71 0.07 2.93 7.06

The diversity of the CREA germplasm (2015-2016)

Caruso et al 2016, Sci Hortic

0

20

40

60

80

100

120

140

160

1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55 57 59 61 63 65 67 69 71 73 75 77 79 81 83 85 87

2015

2016

Average cumulative content of anthocyanin (block 2b)

29/02/2016 24/02/2015

Tota

l anth

ocyanin

(m

g l -

1)

Moro Sanguigno &

Sanguinello Tarocco

The climatic differences

likely affected anthocyanin

accumulation, but not in

the same way for all varieties

-5

5

15

25

-5

5

15

25

335 350 365 15 30 45 60

Tmin Tmax Tmed

75

DOY

°C

Tmax-Tmin

Tmin Tmax Tmed Tmax – Tmin

Average daily minimum, mean and

maximum air temperatures and clines

between December 1st and March 15th.

°C

Air temperatures in the two seasons

Number of hours below 4 °C (15-days

interval) between December 1st and

March 15th.

20

14

-20

15

20

15

-20

16

336-350 351-365 1-15 16-30 31-45 46-60 61-75 0

50

100

150

2014-15 2015-16

DOY

hours

n.

Year-to-year stability of the major traits (germplasm)

ND: not determined

due to lack of

replications;

n.s.: not significant;

statistically

significant (one-way

ANOVA) with the

following p-values:

*<0.05

**<0.01

***<0.001

1. Shamouti 2. Ovale Nuc. 3. Valencia Campbell S2G 18 19 Nuc. 4. Navel Lanelate Nuc.C 2611 5. Navel Cara Cara Lindcove 6. Navel Cara Cara 7. Vaniglia Sanguigno 8. Vaniglia Biondo 9. Tarocco Ferreri acidless 10. Tarocco Dal muso 11. Tarocco Lempso C Nuc. 12. Tarocco TDV 13. Tarocco Ippolito VCR 14. Tarocco Meli C 8158 Nuc. 15. Tarocco Gallo 898 16. Tarocco Scirè D2062 17. Moro non pigmentato Nuc. 18. Moro Nuc. 58 8D 1 Russo 19. Moro VCR 20. Doppio Sanguigno 21. Sanguinello Comune 22. Sanguinello Moscato R I

Requencing of sweet orange genotypes

PCR

Mobile elements HRM

Indels (1-18bp) Sanger & KASP

SNPs

Sanguigno/ Sanguinello

Valencia

Resequenced samples Tarocco

Moro

Vaniglia

Navel

Common

Two old blood orange varieties clustered with the common oranges

Sanguigno tunnuliddu

Vaccaro

Sanguigno Tunnuliddu

Vaccaro

Genotyping of the germplasm collection

Experimental orchard #1

Nurseries #3

Commercial orchards #15

Clonal fingerprinting (190 samples)

Sicily

Basilicata

Sardinia

Sassari

Clonal fingerprinting

Variety Origin SNPs_KASP SVs_PCR

SNP_1 SNP_2 SNP_3 SV_1

Tarocco Meli

Experimental orchard X X X X

Commercial orchard X X X X

Commercial orchard X X X X

Commercial orchard X X X X

Commercial orchard X X X X

Commercial orchard X X X X

Commercial orchard X X X X

Commercial orchard X X X X

Commercial orchard X X X X

Commercial orchard X X X X

Commercial orchard X X X X

Commercial orchard X X X X

Tarocco TDV

Experimental orchard X X / X

Commercial orchard X X / X

Nursery X X / X

Commercial orchard X X / X

Commercial orchard X X / X

Commercial orchard X X / X

Commercial orchard X X / X

The lemon germplasm, and the impact of mal secco disease

Mal secco was first reported in 1918 in Santa Teresa Riva (Messina, Sicily)

Vascular disease caused by the fungus Plenodomus tracheiphilus

Specific symptoms: pink-salmon to reddish discoloration of the wood, desiccation.

Direct damages: heavy yield losses and death of susceptible cultivars

Quarantine pathogen (EPPO list A2)

Phenotyping for mal secco symptoms

25 lemon clonal selections planted in 2002, three replicates

The field trial also included lemon and citron hybrids, autotetraploid lemons and other citrus species as reference.

A few very susceptible clones died within 3 to 5 years after planting, and some others declined slowly without a significant ability to recover from the disease.

Phenotyping for mal secco symptoms started in 2018 on the remaining clones showing variable degrees of tolerance. Three times per year

A molecular screening was also performed to detect possible sources of P. tracheiphilus in leaves and branches

Phenotyping for mal secco symptoms

Sample Symptoms PCR Limone Mascali senza semi (LI) 2 +

lim. Erice su citrumelo (L.E.) 4 +++ lim. Akragas (LAK) 0 +++

lim. Selinunte 0 ++ lim. Zagara Bianca M 79 1 ++

Lim. Segesta 1 +++ Lim. Continella M84 1 ++

Lim. Fem. S 2 ++ Lim. Fem. Dosaco M 503 2 ++

Lim. Kamarina 0 +++ Lim. Cerza 2 +++

lim. Fem Scandurra 2 + Lim. Interdonato 0 ++

Lim. Ovale 2 + Lim. Quattrocchi 0 + Limone Cardinale 0 +

lim. Doppio 0 ++ Doppio lentini (D) 0 -

lim. Lo Porto 0 ++ lim. Adamo variegato 0 -

Berg. Fantastico 0 + Berg. Castagnaro 0 -

Berg. Femminello (incerto) 0 + Pera del commentatore (PC) 0 +

Cedro Vozza Vozza( CVV) 1 + Volkameriana clone catania (VO) 0 -

Lim. Cedrato Spatafora 0 + Lima di Palestina 0 -

Ibrido 46515 3 ++ Limone Incomparabile 0 -

C. limonia (India) CRC 2476 0 ++ lima rossa corrugata 0 +

C. volkameriana clone Palermo 1 lim. India CRC 2322 0 +

Latipes (LA) 0 - Arancio Amaro (AA) 0 -

Siamelo 1 CRC 2586 (SI) 0 + Clementine ISA 0 -

C. tachibana 0 -

LEM

ON

S Le

mo

n a

nd

ci

tro

n h

ybri

ds

Oth

ers 0=no symptoms

1=mild symptoms in one date

2=mild symptoms in more dates

3=strong symptoms in one date

4=strong symptoms in more dates

Thanks for

your

attention!

Personnel Giuseppe Russo Concetta Licciardello Paola Caruso Silvia Di Silvestro Riccardo Russo