Potato (Solanum tuberosum L.), is the third most important crop and the most important non-cereal crop for food security in the world in 2016. (FAO, 2018) The genus Solanum also includes important crops such as, tomato, pepper, aubergine and tobacco along with potato. Potato was domesticated 10,000 years ago in Andean highlands of southern Peru, where landrace potatoes are grown around 3,000 – 4,000 m elevation. (Ovchinnikova et al., 2011, Spooner et al., 2005) Basic chromosome number for potato is 12. Ploidy in potato ranges between diploid to hexaploid, with majority being diploid. The cultivated potato is autotetraploid (2n=4x=48) and due to its high heterozygosity levels, it is vegatatively propagated instead of sexually reproduction. (Bradeen et al., 2016)
Some of the cultivated and wild potatoes produce 2n gametes. Autopolyploidization of these landraces resulted with (S. tuberosum group Andigena; 2n=4x=48) Andean cultivated tetraploids. (Watanabe et al., 1989) Most of the potato cultivars are autotetraploid, heterozygous, suffers from acute inbreeding depression and are vulnerable to pests and pathogens. Many modern cultivars are propagated vegatatively and since they are highly related to each other they are different by only a couple meiotic generations. (Gebhardt et al., 2004, Simko et al., 2004) As a result of these characteristics with narrow genetic base (Love et al., 1999) it is very difficult to improve potato with classical approaches. (PGSC et al 2011) Due to highly heterozygous genome, breeding of potato is a difficulty. It requires a big population of progeny to screen and select the desired individuals. Potato also suffers from inbreeding depression, as a consequence, deleterious mutations occur. (PGSC et al 2011)
DNA Sequencing
— Sanger Sequencing
Recent advances in DNA sequencing and data science make genomic studies more available than ever today as a result of lower costs and faster operations. First DNA sequencing technique was developed by Fred Sanger and Alan Coulson in 1977. (Sanger et al., 1977) Sanger sequencing exploits the use of dideoxynucleosides as chain terminators. In this technique a primer binds to the DNA strand of interest, with DNA polymerase enzyme deoxyribonucleotide triphosphates(dNTP’s) and dideoxynucleosides (ddNTP’s) polymerize on the many copies of the DNA strand. This reaction results with DNA strands with different length, all of them terminated by ddNTP’s. Separating the strands according to their length with gel electrophoresis and determination of the ddNTP’s of each length reveals the DNA sequence. (Sanger et al., 1977)
Figure 1: Sanger sequencing. (Brown et al., 2002)
— Pyrosequencing
Faster and better DNA sequencing methods are evolving every day. Pyrosequencing is another DNA sequencing method. This technique doesn’t include ddNTP’s, sequencing is being done right when a dNTP added to the DNA strand. When dNTP is added to the DNA strand it releases pyrophosphate which then converted into light that is detected by sensors. dNTP’s quickly desaturate after presentation to the medium and washed away thus with the known dNTP addition one after another DNA sequence can be detected. (Ronaghi et al., 1998) This method quickly evolved and in 2005, 454 Life Sciences Corp. established a new method using pyrosequencing. (Margulies et al., 2005) And thus Next Generation Sequencing (NGS) era began.
— Library generation
—Nest Gen Seq Details
In NGS technology first step is to produce a library of the DNA that’ll be sequenced. This step consists of ligation of special DNA sequences (adapters) at the ends of the DNA strands. Then adapter added DNA library fragments are amplified in situ which means on a solid surface.
– Short / Long reads
– Seq by ligation
– Seq by synthesis
—- Map Generation
– Genetic
– Physical
—-Assembly methods
– Shotgun
– Clone
—Plant Seq History
—Potato reference genome #1
De novo assembly of a genome is long and sophisticated process. Having a reference genome to map the sequence onto makes the process fairly easier. First, Potato Refence Genome has been published in 2011 by Potato Genome Sequencing Consortium. (PGSC et al., 2011)
In this study, to overcome the difficulties highly heterozygous diploid potato genome bares, homozygous double monoploid of S. tuberosum group Phureja DM1-3 516 R44 (DM) is used. S. tuberosum group Tuberosum RH89-039-16 (RH), a heterozygous diploid cultivar, was sequenced and used to map onto the anchored DM genome.
Genomic DNA from DM was sequenced using Whole-Genome shotgun sequencing approach with Sanger, Illumina Genome Analyzer 2 (GA2) and Roche 454 platforms. BAC library and fosmid libraries were constructed using Sanger platform. To assemble the whole genome Illumina GA2 pair-end short reads were assembled into contigs using SOAPdenovo short read assembly software. To generate scaffolds from contigs, pair-end relationships, mate-pair reads, fosmid ends and BAC ends were used. Then gaps further filled using 454 data. DNA sequencing of the RH was done on Illumina GA2 and 454 platforms. Anchoring of the contigs onto chromosomes done with a de novo developed genetic map. This map was consisted of sequence-tagged-sites (STS), simple sequence repeats (SSR), SNP’s and diversity array technology (DArT). Two approaches were taken during anchoring of the genome. First approach used genetic map to anchor the contigs and second approach used RH ultra-high-density linkage map. AFLP’s of RH genetic map was linked to DM with BLAST alignment. Overall their assembly was 727 Mb (93.9% non-gapped) which is 117 Mb less than the estimated genome size of 844 Mb. (Bennet et al., 1997) They found that 62.2% of this assembled genome is repetitive sequences while 29.4% is transposable elements. They were able to anchor 86% of the assembled genome which includes 90.3% of the predicted genes number (39,031). (PGSC et al., 2011)
2 years after the first potato reference genome Sharma and his colleagues published the newer version of the first potato reference genome. (Sharma et al., 2013)
— Wild Specie Potato Genome (Aversano 2015)
— Potato Reference Genome #2 (Lesiner, 2018)
— CNV analysis