Development of a Mitochondrial SCAR Marker Related to Susceptibility of Banana ( Musa AAA Cavendish) to Fusarium oxysporum f. sp. Cubense Race 4

The use of resistant cultivars is an effective method for the control of banana ( Musa spp.) Fusarium wilt caused by race 4 of Fusarium oxysporum f. sp. cubense ( Foc 4). However, selection of disease-resistant cultivars requires large-scale field evaluations and is time-consuming. Development of early, reliable, and reproducible selection strategies can speed up this process. Sequence characterized amplified region (SCAR) markers have been widely employed in the resistant breeding of many crops. However, to date, there have been no reports about the presence of plant disease resistance-related SCAR markers in mitochondrial genome yet, which also plays a very important role in plant defenses. In the present study, a sequence-related amplified polymorphism (SRAP) marker, a specific fragment of 829 bp, was identified. This fragment could be amplified from Foc 4-susceptible but not from the resistant cultivars. It was located in banana mitochondrial genome and mapped near the putative cytochrome c biogenesis ccmB-like mitochondrial protein. This fragment was then successfully converted into a SCAR marker, namely Mito-Foc-S001, which was found to be able to discriminate the resistance from susceptibility to Fusarium wilt disease of bananas with the discriminatory power of the new mark being 96.88%. Thus, this marker can be used in banana ( Musa AAA Cavendish) breeding for Fusarium wilt disease resistance.


Introduction
Banana and plantain (Musa spp.) are the fourth and the second most important food and fruit crop in the world, respectively (FAO, 2017). Banana and plantain are also the most important fresh fruit in international trade market in terms of production and consumption volume (FAO, 2013). Banana Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense (Foc), is one of the most devastating diseases, especially in Asia and Africa. Foc has been classified into four physiological races according to the symptoms of different banana cultivars (Pegg et al., 1996;Bentley et al., 1998). Among them, Foc race 4 (Foc4) is the most virulent one (Sun et al., 2010). This race was first reported in Taiwan in 1969 (Sun et al., 1978) and subsequently spread to Australia, Africa and a number of Asian countries (Hwang and Ko, 2004). In China, banana Fusarium wilt caused by Foc4 has been successively reported in Guangdong, Guangxi, Fujian, Hainan provinces, including nearly all the main banana-production areas (Li et al., 2011). To date, this disease has become the key factor seriously limiting banana production in China (You et al., 2008). Since Fusarium wilt is a soil-borne disease, there has been no chemical control approach available and the only way to control it is to plant the cultivars resistant/tolerant to this pathogen (Ploetz, 2006). resistant germplasm and facilitate molecular marker-assisted selection of novel banana cultivars resistant to this pathogen.

Plant material
A total of thirty-two banana (Musa AAA Cavendish) cultivars with different degrees of resistance or susceptibility to Foc4 were used in the present study and their characteristics were presented in Table 1. The test of their resistance or susceptibility was carried out in the inoculated field for identification of resistant banana germplasm after a 3-year period (from 2012-2015) of observation and investigation.
DNA extraction DNA was extracted by a modified cetyltrimethyl ammonium bromide (CTAB) method described by Javed et al. (2004) with modifications. In brief, fresh leaves (0.2 g) were ground to powder in liquid nitrogen with cross-linked polyvinyl pyrrolidone (PVP), suspended in 800 μL of preheated extraction buffer [4% CTAB, 1.4 M NaCl, 20 mM ethylene diamine tetraacetic acid (EDTA), 100 mM Tris-HCl [pH 8.0] and 1% 2-mercaptoethanol at 65 °C for 30 min, followed by centrifugation at 12000 rpm for 10 min. The supernatant was saved and extracted twice with equal volumes of chloroform/isoamyl alcohol (24:1). The supernatant was combined and centrifuged at 12000 rpm for 6 min. DNA was pelleted with equal volumes of precooled isopropanol (-20 °C). After being rinsed with 75% pre-cooled ethanol, DNA sample was dried under vacuum and then dissolved in 50 μL of TE buffer [10 mM Tris-Hcl (pH 8.0), 1 mM EDTA, pH 8.0]. The DNA concentration was determined with an ultra low volume spectrometer (BioDrop μLite, Biochrom/UK). The concentration was adjusted to 50 ng.μL -1 with ddH2O before use.
PCR amplifications were performed according to the manufacturers' instructions and run in a Bio-Rad PCR (Bio-Rad T100, Bio-Rad, Hercules, CA, USA) with a total reaction mixture of 25 μL, containing 2.5 μL of 10×buffer (with Mg 2+ ), 2μL of 0.25 mM dNTPs, reverse and inverse primers at a final concentration of 0.4 μM, 1 μL of DNA template (50 ng.μL -1 ) and 1.5 unit of TaqDNA polymerase (Tiangen, Beijing, China). Reactions were programmed to an initial denaturation for 5 min at 94 °C, followed by 5 cycles as follows: 1 min at 94 °C denaturation, 1 min at 35 °C annealing and 1 min at 72 °C elongation and then by 35 cycles consisting of 1 min at 94 °C denaturing, 1 min at 50 °C annealing and 1 min at 72 °C elongation, and final elongation at 72 °C for 8 min.
Conventional banana breeding is mainly hampered by the fact that most cultivars are triploid, parthenocarpy and thereafter, sterility. Biotechnological methods can contribute significantly to the genetic improvement of agronomic traits of banana cultivars, including disease resistance (Xu et al., 2011;Kovács et al., 2013;Umber et al., 2016). However, selection of disease-resistant somaclones requires large-scale field evaluations and is time-consuming (Javed et al., 2004) whereas sometimes the susceptible plants have a chance to escape detection (Burger et al., 2003) due to various other factors. Thus, development of early, reliable, and reproducible selection strategies can speed up the selection procedure and, eventually, improvement of banana production and quality (Javed and Othman, 2005).
However, all the reported SCAR markers linked to plant resistance are located in nuclear chromosomes. Mitochondria are one of the most plant cell organelles and have been suggested to play a very important role in plant defenses (Gleason et al., 2011;Laluk et al., 2011;Ambrosio et al., 2013;Qamar et al., 2015;Fuchs et al., 2016). Furthermore, mitochondrial DNA (mtDNA) is also widely used as molecular markers in the fields of systematic evolution, cytoplasmic male sterility and so on (Ji et al., 2014;Gao et al., 2015;Shu et al., 2016). However, to date, there have been no reports about the presence of plant disease resistance or susceptibility-related SCAR markers in mitochondrial genome yet. Whether SCAR markers related to plant disease susceptibility or resistance are present in mitochondrial genome remains to be determined.
In the present study, we developed a novel SCAR marker, which was located in mitochondrial genome and associated with Foc4 susceptibility of Musa AAA Cavendish, through sequence-related amplified polymorphism (SRAP)-based screening. This marker could discriminate the resistance from susceptibility to Fusarium wilt disease of bananas with the discriminatory power of the new mark being 96.88%. The availability of this novel SCAR marker will provide an essential tool to diagnose Foc 510 5′-TGAGTCCAAACCGGAAT-3′ em10 5′-GACTGCGTACGAATTCAT-3′ SRAP: sequence-related amplified polymorphism.
After amplification, 4 uL of 6 × loading buffer was added to the PCR amplified products. 8-10 μL of PCR products were fractionated by gel electrophoresis on 1.8% (w/v) agarose gels in 0.5 × Tris-borate-EDTA (TBE) buffer (pH 8.0). Gel was run at 120 V for 1 h, stained with ethidium bromide, visualized on a UV transilluminator and photographed with gel imaging system (UVI FireReader, UK).
The amplified products of the linked SRAP were excised from agarose gels, and the DNA was purified by Tiangel Midi purification kit (Tiangen) according to the manufacturers' instructions followed by sending to Thermo Fisher (China) Co., LTD. Guangzhou Branch for sequencing. The purified products was amplified with BigDye® Terminator v3.1 Cycle Sequencing Kit [Thermo Fisher Scientific, Applied Biosystems, Foster City, CA, USA], and the PCR products were purified using the Mag-Bind® RxnPure Plus (OmegaBiotek, Norcross, GA, USA). Finally, the products were sequenced using a 3730xl DNA analyzer [Thermo Fisher Scientific (Applied Biosystems)].

Validation of the SCAR marker
The linked SCAR marker obtained in the present study was validated with another 24 banana cultivars with different degrees of resistance to Foc4 (cultivars No. 9-32 in Table 1), including the two cultivars originally used as the templates of SRAP. PCR reaction system was referred to the method described in 1.4.

DNA quality
The quality of the extracted DNA was checked with BioDrop μLite (Biochrom/UK). The concentration of DNA samples was between 80 and 150 ng.μL -1 , the OD260/OD280 ratios of all the samples were in the range of 1.8 to 2.0, indicating that the quality is good enough to be used for further experiment.

Screening of SRAP molecular markers involved in disease resistance
As shown in Fig. 1, a special band of 839 bp was amplified from DNA templates derived from three susceptible cultivars ('Baxijiao', 'NK4-1', and 'L3-3') by PCR with primer me6-em1 out of 100 pairs of SRAP primers tested. However, it was absent in all the resistant cultivars.

Development of a SCAR marker involved in banana resistance to Foc4
The PCR amplified products were collected from cultivars 'Baxijiao', 'NK4-1', and 'L3-3'. The qualities and concentrations were good enough. These products were sequenced and compared with that of MAGE6.0. The result showed that their sequences were exactly the same.  No assignment to a particular chromosome or resemblance with known resistance or susceptibility genes was revealed when blasted this fragment in NCBI. However, when being compared with Banana Genome Hub database (http://banana-genome-hub.southgreen.fr/blast), it showed a 99.64% similarity to a fragment located in mito4 of the M. acuminata spp. malaccensis accession Pahang (2n = 22) genome (start from the third nucleotide). This fragment is located between nucleotides 145, 680 and 146, 506 and mapped near the putative cytochrome c biogenesis ccmBlike mitochondrial protein.
To develop SCAR markers, we selected four pairs of primers presented in Table 3 to amplify the PCR products from 8 banana cultivars with different degrees of resistance to Foc4. Fig. 2 showed that a single and clear band of 324 bp was amplified with primers SC1 and SC2 and appeared only in five susceptible cultivars ('Baxijiao', 'NK4-1', 'L1N2', 'L3-3', and 'WS2') but not in the three resistant ones ('Nantianqing', 'Dongjiao 1', and 'Kangku 1'). This fragment is located in the position from 216 to 539 bp of the original SRAP band (located between the mitochondrial genome nucleotides 145, 894 and 146, 217). The quality of the band was good enough to serve as a SCAR marker and thus, was considered a suitable candidate for the development of the SCAR marker, and finally selected. On the other hand, some other special bands were also obtained with the other three pairs of primers, which also could distinguish the resistant cultivars from the susceptible ones; however, their qualities were not good enough to be used as a SCAR marker (data not shown).

Discussion
As mentioned above, banana Fusarium wilt is one of the most devastating diseases found in a large majority of the banana-producing regions in the world except the Mediterranean, Melanesia, Somalia, and some islands in the South Pacific (Ploetz, 2006). To date, no methods for efficient control of this disease are available. The best way to control this disease is to plant the resistant cultivars. Identification of molecular markers linked to disease resistance is an excellent alternative to enable banana breeders to make marker-assisted selection for disease resistance without challenging the host with the pathogen. Javed and Othman (2005) studied the resistance of Musa acuminata to Fusarium oxysporum using the technique of RAPD. The SCAR markers associated with the resistance to this disease was first reported by Wang et al. (2012). They detected the resistance of clones from 'Williams' (Musa spp. AAA) using two SCAR markers, namely ScaU1001 and ScaS0901, which were amplified in 'Gold finger' (Musa spp. AAAB) and one clone from 'Williams'. These SCAR markers could not be detected in 5 susceptible cultivars tested. Recently, Cunha et al. (2015) generated a SCAR marker from RAPD markers which could identify susceptibility to Fusarium oxysporum f. sp. cubense for some banana cultivars belonging deferent genotypes in South Brazil. They validated this SCAR marker on 28 banana cultivars resistant or susceptible to infection by Foc. Their result showed that this new marker could discriminate two contrasting groups from each other with the discriminatory power of 93%.
In the present study, we identified a novel and specific SCAR marker from 100 pairs of SRAP primers associated with susceptibility to Foc4. We tested the specificity of this marker against a total of 32 banana cultivars exhibiting different degrees of resistance or susceptibility to Foc4. Nine resistant cultivars including the most resistant germplasm in China and a few from other countries and 23 susceptible cultivars including the main cultivars grown in China and some other countries as well as a number of the local cultivars in China were purposely selected, respectively. The new SCAR marker, namely Mito-Foc-S001, could distinguish the resistant group from the susceptible one with the discriminatory power of 96.88%. The sequence of PCR product from primers SC1 and SC2 had high homology with a fragment in mito4 of banana, indicating that this SRAP-based SCAR marker is a gene located in the mitochondrial genome instead of in the nuclear chromosomes. Though no a particular chromosome or resemblance with known resistance or susceptibility genes have been assigned with the fragment identified in this study, further developments may enable us to reveal similarities of this sequence to the known genes.
Mitochondria are semi-autonomous organelles that possess their own genomes and are able to synthesize a small number of proteins essential for the functions of mitochondrial respiratory chain. They are the main fuel stations of eukaryotic cells because they are a major source of ATP. In addition, they are vital for cellular homeostasis, participating in diverse metabolic pathways, calcium signalling and reactive oxygen species buffering or production. These functions link mitochondrial activities to the environment and with other cellular compartments such as chloroplast (Welchen et al., 2014). Many earlier reports revealed that mitochondria contributed substantially to responses to plant stress and defense responses (Gleason et al., 2011;Laluk et al., 2011;Ambrosio et al., 2013;Qamar et al., 2015;Fuchs et al., 2016) and/or play a fundamental role in innate immunity signalling (Vellosillo et al., 2013). Defects in mitochondrial functions could increase susceptibility of plants to pathogens (Pu et al., 2016). Thus, it was suggested that the SCAR marker obtained in the present study also likely plays a very important role in banana susceptibility to Foc4. However, no assignment to a particular chromosome or resemblance with known resistance of susceptibility genes was revealed. The information about this novel marker is quite limited. Further work should be focused on acquiring these pieces of information and investigating the specific functions of mitochondrial genes in banana defenses.

Conclusions
Our results demonstrate that the SCAR marker Mito-Foc-S001, could discriminate the resistance from susceptibility to Fusarium wilt disease of bananas with the discriminatory power of the new marker being 96.88%. The application of the marker is simple and efficient, and has a great advantage for the screening of large number of banana individuals, since only DNA extracted and a simple PCR are required. Thus, this marker can be used in banana (Musa AAA Cavendish) breeding for Fusarium wilt disease resistance.