**2.4. Functional annotation**

**1. Introduction**

46 Advances in Seed Biology

Avocado (*Persea americana* Mill.) is an oleaginous fruit produced by a tree belonging to the magnoliid clade, a basal linage of flowering plants. It belongs to the large plant family of Lauraceae, with approximately 2500–3000 species [1, 2]. Avocado has been rapidly incorporated as a component of human diet in many countries [3]. Due to low cost, vigor of seedling growth and easy propagation, most of the countries are still using seeds to produce rootstocks for grafted avocado trees despite their genetic variability [4]. Several Mexican varieties are derived from seeds that are resistant to attack by *Phytophthora cinnamomi* [5, 6] and are adapted to the soil and environmental conditions of the region. The Mexican state of Michoacán is the primary avocado-producing region in the world, and all the rootstocks used for the commercial production of cultivar Hass are obtained from *P. americana* var. drymifolia ("nativo mexicano") [7]. The principal consume form is as fresh fruit, but is really important in cosmetic industry [8]. Avocado plant has medicinal properties, including cancer prevention [9–11]. There is ethnopharmacological information on the use of avocado seeds for the treatment of health-related conditions, especially in America. Recent research has shown that the avocado seeds are rich in phenolic compounds and these maybe play a role in putative health effects [12]. The avocado fruit is a berry of one carpel containing a single seed. This large and very conspicuous seed is made up of two fleshy cotyledons and a central attached plumule, hypocotyl and radicle, the whole surrounded by two papery seed coats closely adherent to each other. There is no endosperm left in the seed at maturity. The cotyledons are formed of indifferentiated parenchyma tissue interspersed with occasional idioblasts. Starch is the main storage material of the cotyledons and is present in great abundance [13]. Despite its importance, avocado seed development remains uncharacterized. To date, little information is available regarding the molecular biology of the seed. Analysis of expressed sequence tags (ESTs) is a rapid and effective method to identify novel genes or to investigate gene expression in different tissues, organs and plants [14, 15]. Furthermore, EST libraries and databases could provide valuable resources for functional genomic studies [16].

In principle, the frequency with which the sequence of a given gene is read in ESTs sequencing projects should reflect the relative abundance of the corresponding mRNA. This approach

In this work, we report the analysis of an ESTs collection from immature avocado nativo mexicano seeds and the analysis of expression of bHLH transcription factor, metallothioneins

Seeds from avocado nativo mexicano fruits of three stages of development (1, 4 and 8 months) were excised from fruits and frozen immediately in liquid nitrogen until use. The materials

uses EST counts to infer the relative level of expression of a gene [17–19].

(MTs) and snakin like more abundantly expre3ssed.

**2. Materials and methods**

**2.1. Biological material**

Stand-alone BLAST software was obtained from the National Center for Biotechnology Information (NCBI, http://www.ncbi.nlm.nih.gov). The unigenes were compared by BLAST against nucleotides and proteins plant databases. The BLAST results from different databases were used for gene ontology (GO) mapping and annotation. Blast2Go software was used to perform GO functional classification.
