Determination of Self- and Inter-(in)compatibility Relationships in Apricot Combining Hand-Pollination, Microscopy and Genetic Analyses

Summary

We present a methodology to establish the pollination requirements of apricot (Prunus armeniaca L.) cultivars combining the determination of self-(in)compatibility by fluorescence microscopy with the identification of the S-genotype by PCR analysis.

Abstract

Self-incompatibility in Rosaceae is determined by a Gametophytic Self-Incompatibility System (GSI) that is mainly controlled by the multiallelic locus S. In apricot, the determination of self- and inter-(in)compatibility relationships is increasingly important, since the release of an important number of new cultivars has resulted in the increase of cultivars with unknown pollination requirements. Here, we describe a methodology that combines the determination of self-(in)compatibility by hand-pollinations and microscopy with the identification of the S-genotype by PCR analysis. For self-(in)compatibility determination, flowers at balloon stage from each cultivar were collected in the field, hand-pollinated in the laboratory, fixed, and stained with aniline blue for the observation of pollen tube behavior under the fluorescence microscopy. For the establishment of incompatibility relationships between cultivars, DNA from each cultivar was extracted from young leaves and S-alleles were identified by PCR. This approach allows establishing incompatibility groups and elucidate incompatibility relationships between cultivars, which provides a valuable information to choose suitable pollinizers in the design of new orchards and to select appropriate parents in breeding programs.

Introduction

Self-incompatibility is a strategy of flowering plants to prevent self-pollination and promote outcrossing¹. In Rosaceae, this mechanism is determined by a Gametophytic Self-Incompatibility System (GSI) that is mainly controlled by the multiallelic locus S². In the style, the RNase gene encodes the S-stylar determinant, a RNase³, while a F-box protein, which determines the S-pollen determinant, is codified by the SFB gene⁴. The self-incompatibility interaction takes place through the inhibition of pollen tube growth along the sty....

Protocol

1. Self-(in)compatibility determination

1. Sample the flowers in the field. It is necessary to collect the flowers at balloon stage (Figure 1A), corresponding to stage 58 on the BBCH scale for apricot²⁸, to avoid unwanted previous pollination.
2. Self- and cross-pollinations in the laboratory
   1. Remove the anthers of the flowers at balloon stage and place them on a piece of paper to dry at laboratory temperatu.......

Discussion

Traditionally, most commercial apricot European cultivars were self-compatible³⁶. Nevertheless, the use of North American self-incompatible cultivars as parents in breeding programs in the last decades has resulted in the release of an increasing number of new self-incompatible cultivars with unknown pollination requirements^7,8,37. Thus, the determination of self- and inter-(in)compatibility relationships.......

Materials

Name	Company	Catalog Number	Comments
Agarose D1 Low EEO	Conda	8010.22
BIOTAQ DNA Polymerase kit	Bioline	BIO-21060
Bright field microscope	Leica Microsystems	DM2500
CEQ System Software	Beckman Coulter
DNeasy Plant Mini Kit	QIAGEN	69106
dNTP Set, 4 x 25 µmol	Bioline	BIO-39025
GenomeLab DNA Size Standard Kit - 400	Beckman Coulter	608098
GenomeLab GeXP Genetic Analysis System	Beckman Coulter
GenomeLab Separation Buffer	Beckman Coulter	608012
GenomeLab Separation Gel LPA-1	Beckman Coulter	391438
HyperLadder 100bp	Bioline	BIO-33029
HyperLadder 1kb	Bioline	BIO-33025
Image Analysis System	Leica Microsystems
Molecular Imager VersaDoc MP 4000 system	Bio-Rad	170-8640
NanoDrop One Spectrophotometer	Thermo Fisher Scientific	13-400-518
pH-Meter BASIC 20	Crison
Phusion High-Fidelity PCR Kit	Thermo Fisher Scientific	F553S
Power Pack P 25 T	Biometra
Primer Forward	Isogen Life Science
Primer Reverse	Isogen Life Science
Quantity One Software	Bio-Rad
Stereoscopic microscope	Leica Microsystems	MZ-16
Sub-Cell GT	Bio-Rad
SYBR Safe DNA Gel Stain	Thermo Fisher Scientific	S33102
T100 Thermal Cycler	Bio-Rad	1861096
Taq DNA Polymerase	QIAGEN	201203
Vertical Stand Autoclave	JP Selecta