Evaluation of genetic diversity in self-incompatible broccoli DH lines assessed by SRAP markers

In this article, we investigated self-compatibility index (SCI) in broccoli double haploid (DH) lines, and the relationship and genetic diversity of 15 self-incompatible (SI) broccoli DH lines were analyzed by sequence related amplified polymorphism (SRAP). 11 primer combinations selected from 88 primer pairs revealed a total number of 129 unambiguous bands, 61 of which were polymorphic with a polymorphism frequency of 47.3%. Analyzed by NTSYS software, the genetic similarity coefficient of the 15 broccoli resources ranged from 0.76 to 0.98. Based on the coefficient value of 0.79, these broccoli DH lines were clustered into three multiple-member groups by unweighted pair group method with arithmetic mean (UPGMA) analysis, which provided molecular reference for parent selection in broccoli breeding.


INTRODUCTION.
Broccoli (Brassica oleracea L. var.italica) is a traditional European crop, and now has become widespread in Asian in recent decades (Branca, 2008).In broccoli, F 1 hybrids have advantages especially in uniform maturity, high total yield, better curd/head quality, and resistance to diseases and unfavourable weather conditions (Branca, 2008).For producing hybrid seeds of cabbage, cauliflower, broccoli, Brussels sprout and kale, a selfincompatibility (SI) character is utilized which is controlled by the S-locus (King, 2003).For establishing the homozygous SI parental lines, it usually needs years to *Corresponding author.E-mail: gu2199@yahoo.com.cn.Tel: +86-571-86417316.
Abbreviations: SCI, Self-compatibility index; DH, double haploid; SRAP, sequence related amplified polymorphism; UPGMA, unweighted pair group method with arithmetic mean; SI, self-incompatible; SSI, sporophytic self-incompatibility; SRK, S receptor kinase; SP11/SCR, small cysteine-rich secreted protein; get inbred line, survey and choose for several generations.Microspores culture could fleetly get homozygous lines-double haploid) (DH) lines and surveying self-compatibility indexes (SCI) of DH lines could quicken the getting of homozygous SI lines.Broccoli belongs to Brassicaceae.In the Brassicaceae, a conserved sporophytic self-incompatibility (SSI) system is present, and detailed genetic studies have resulted in the identification of highly polymorphic S genes that confer this trait.
The SSI system has been best characterized in the genus Brassica, and is primarily controlled by a receptorligand system encoded in two tightly linked and multiallelic genes: the S receptor kinase (SRK), and the small cysteine-rich secreted protein, SP11/SCR (Samuel et al., 2008).SRK is the sole determinant of specificity in the stigma, and encodes a membrane-associated receptor protein kinase with extracellular, transmembrane and cytoplasmic kinase domains (Takasaki et al., 2000;Silva et al., 2001).SP11/SCR is the male determinant of Slocus specificity in the pollen side and encodes a lowmolecular weight cysteine-rich protein which specifically expresses in the anther tissues (Shiba et al., 2001).The co-evolved SRK and SP11/SCR alleles constitute different S-haplotypes, and 'self' pollen rejection occurs
Although the interactions between SRK and SP11/ SCR has been well mapped out, still there are several questions to be solved, such as how temperature and humidity have impact on the SI, and how to overcome SI during reproduction of SI plants.Research on broccoli SI is seldom.In this article, we investigated SCI in broccoli DH lines, and evaluated genetic diversity of SI materials in broccoli by sequence related amplified polymorphism (SRAP).

MATERIALS AND METHODS
DH lines were planted in conservatory in 2008 autumn and SCI were determined in the next year spring when plants flowered.

Determination of self-compatibility index
Before buds anthesis, florets were protected from other pollens pollinated by bags.When most buds of the florets flowered, the bags were wiped off and flowers were pollinated with the pollen of the same plant and they were protected from other pollens for about 1 week after pollination.In every DH lines, 50 flowers were pollinated in early flower (in March).All other flowers or florets were discarded and the indication of plant name was written on a tag.SCI was determined again when plants were in the final-phase flower (in April).

DNA extraction
According to the result of determination of SCI, tender leaves of low SCI plants were taken for genomic deoxyribonucleic acid (DNA) isolation according to a cetyl trimethylammonium bromide (CTAB) procedure (Li and Quiros, 2001).

SRAP analysis
In this assay, exceptional SRAP primers (me6-me8, em7-em11) were designed except those mentioned in Li and Quiros' paper.All the primers were commercially synthesized (Sangon biological engineering technology and service Co. LTD., Shanghai).A total of 88 different combinations were employed using eight forward primers and 11 reverse primers (Table 1).Polymerase chain reaction (PCR) amplification was according to the procedure of Li and Quiros (2001).

Scoring and data analysis
The PCR products from SRAP analyses were scored qualitatively for presence or absence of bands.Only clear and apparently unambiguous bands were scored for SRAP.Genetic similarities between the SI DH lines were measured by the Dice similarity coefficient based on the proportion of shared alleles using 'simqual' sub-program of software NTSYS-PC version 1.8 (Exeter Software, Setauket, NY, U.S.A.) software package (Rohlf, 1993).The resultant distance matrix data was used to construct dendrograms by using the un-weighted pair-group method with an arithmetic average (UPGMA) subprogram of NTSYS-PC (Rohlf, 1993).

Self-compatibility indexes of broccoli DH lines tested
SCI of 124 broccoli DH lines were determined (Table 2).Self-compatible (SC) broccoli DH lines existed, but most of the broccoli DH lines were SI.Out of 124 broccoli DH lines, SCI of 15 lines were greater than 1, that is selfcompatible, and 105 lines SI. b08266, b08267, b08268, b08269, four broccoli DH lines' SCI s were different hugely in the two SCIs tests.Out of 105 invariable (Tables 2 and 3).originates from the variation of the length of introns, promoters and spacers, both among individuals and among species.In genetic diversity analysis, the given by SRAP markers was more concordant to the morphological variability and to the evolutionary history of the morphotypes than that of the amplified fragment length polymorphism (AFLP) markers (Ferriol et al., 2003).In the SRAP analysis, the number of amplified fragments varied from 8 to17, with an average of 11.7±3.07bands per primer combination, and the polymorphism percentage averaged to 47.3% across all the varieties.SRAP analysis was successful in detecting genetic diversity and relationships between the broccoli SI DH lines.A low level of genetic diversity was found in the broccoli SI germplasm.In cluster analysis, 2209-13, 2209T1 and 2209T37, which were from Li lv F 4 inbred line, were highly similar (genetic similarity greater than 0.95).2246T and 2246T1 from the same variety F 1 were less similar, and were clustered into two different groups.That is to say, genetic background of donor decides genetic relationship among donor's DH lines; the more miscellaneous genetic background of donor is, the more complex genetic relationship among donor's DH lines are.Contrarily, the more simplex genetic background of donor is, the lower genetic diversity among the donor's DH lines.

Figure 1 .
Figure 1.Dendrogram of the 15 broccoli self-incompatible DH lines based on SRAP bands using UPGMA cluster analysis.

Table 4 .
Data on SRAP fragments and polymorphism obtained using 11 primer combinations in 15 broccoli DH lines.