Advances in Studies of Genetic Improvement of Sugarcane

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Guangzhou Sugarcane Industry Research Institute/Guangdong Key Lab of Sugarcane Improvement & Biorefinery, Guangzhou 510316, China

1 Introduction

Sugarcane (Saccharumspp.) is the largest sugarcane crop in China and the world. Sucrose accounts for more than 75% of the total sugar yield of the world, and more than 90% of China’s sugar yield. Sugarcane is also the first generation bioenergy crop, and it can be used as renewable energy for production of fuel ethanol and biomass products. From the 1970s, many countries started formulating the clean energy strategy of developing fuel ethanol to replace oil. Among these countries, Brazil was most successful in using sugarcane to produce fuel ethanol as power energy of automobile. In 1979, the first bioenergy automobile using fuel ethanol as drive appeared. By 2003, there were 15.5 million cars using mixed fuel of ethanol and oil, and there were 2.2 million cars completely using ethanol as the fuel[1]. In 2010, the yield of bioenergy using sugarcane as main material had replaced about 47.6% energy consumption of Brazil, bringing it to be the country with highest bioenergy utilization rate[2]. Sugarcane is the generally recognized C4 plant with high photosynthetic rate and dry matter accumulation ability[3-4], and is the crop with highest per unit are yield up to one ton[5]. According to statistics of the Food and Agriculture Organization (FAO), in 2011/2012 sugarcane crushing season, the total sugarcane yield of Brazil reached 7.34×108t, with the per unit area yield up to 76.45 t/hm2. In India and China, the planting area, total yield, and average yield of sugarcane was 4.94×106hm2, 3.42×108t, and 69.25 t/hm2, 1.73×106hm2, 1.15×108t, 66.52 t/hm2separately.

2 Target of sugarcane genetic improvement

Sugarcane is an essential sugar crop and energy crop, and its variety improvement is receiving close attention. Sugarcane has unique genetic mode. It is allopolyploid crop generated through a series of hybridization of polyploidSaccharumofficinarumL. (2n= 80,X= 10) as female parent andSaccharumspontaneumL. (2n= 40～128,X= 8) as male parent. The number of chromosomes is 100-150, about 75%-85% fromSaccharumofficinarumL., and 15%-25% fromSaccharumspontaneumL. In the first and second time of hybridization process, chromosomes were delivered in 2n+nspecial manner. Agronomic characters such as sugar content and yield of hybrid generation were rapidly recovered and stabilized. This process is called nobility process of sugarcane[7]. However, such unique genetic mode increases the difficulty in sugarcane genetic improvement. With rapid development of modern biotechnology, and drop of genome sequencing costs, the development of sugarcane functional genomics and structural genomics plays a great role in promoting further development of sugarcane genetic improvement. Variety is the key for development of the sugarcane industry. Fine variety not only increases the per unit area yield and sucrose content, reduces production costs, but also extends the crushing period, increases equipment utilization rate of sugar factory, and obtains higher economic benefits. With reference to sugarcane production and development in China and foreign countries, it is necessary to constantly upgrade varieties, to promote sustainable and healthy development of sugarcane industry and satisfy the market demands. According to requirements of modern sugarcane industry, the sugarcane breeding targets can be classified into two types: (i) improvement of variety traits, including improving sugar content (sucrose content, brix, apparent purity, and gravity purity), increasing yield (tillering capacity, effective stalk number, stalk diameter, plant height, growth rate, sprouting rate, and ratoon performance), increasing stress resistance (disease resistance, insect resistance, lodging resistance, drought resistance, cold resistance and barren resistance), and selecting varieties (sugarcane hair and defoliation)[8-9]; (ii) cultivation of different purposes of varieties, mainly including sugar type sugarcane (fiber fraction ≤14%), energy type sugarcane (fiber fraction ≥30%), and sugar energy type sugarcane (fiber fraction < 30%)[10].

3 Advances in conventional breeding of sugarcane

3.1DevelopmentstagesofsugarcanebreedingThe development of sugarcane breeding can be divided into 5 stages[9]. (i) The stage of breeding using tropical strain. This stage started from 1858 when Barbados reported that sugarcane can bear fruit. At this stage, main characteristics of varieties included high sugar content, low fiber fraction, high purity, low adaptation, weak ratoon, and poor disease resistance, such as H109, B716, and Q813,etc[11]. (ii) The stage of breeding using nobility process to select noble varieties. In 1885, Soltwedel made experiment of hybridization between sugarcane and Erianthus arundinaceus[12]; in 1893, Moquette and Wakker obtained hybrid variety of tropical strain Black Cheribon and Indian strain Gansha[13]; in 1897, Kobus hybridized Indian strain Chunni and tropical strain[14]; in 1911, Wilbrink successfully hybridized Indian strain Gansha and tropical strain[15]; later, Jeswiet backcrossed the tropical strain with the hybrid generation obtained by Wilbrink, rapidly recovered and stabilized sugar content and yield of hybrid generation[16]; in 1921, Jeswiet successfully bred POJ2878 fine variety through hybridization[17]. This stage opened the curtain of interspecific hybridization, and recovered and stabilized sugar content and yield of hybrid generation, and bred excellent varieties with high sugar content, high fiber fraction, high adaptation, strong ratoon, and high disease resistance, such as Co281 and Co290[18]. (iii) The stage of breeding using noble varieties. From 1930 to 1950, it was the stage of hybridization using noble varieties to select excellent new varieties. Typical examples included Co419 bred using POJ2878 × Co290 in 1937, popular NCo310 in the 1950s and 1960s bred in 1939, and H32-8560 bred by Hawaii Research Center in 1945 (accounting for more than 60% of the local planting area)[17]. This stage mainly used noble varieties to hybridize and breed more germplasm resources. (iv) The stage of breeding using noble hybrid varieties. From 1950 to 1965, noble hybrid varieties were used to select modern sugarcane varieties with high sugar content, high yield, high stress resistance, and excellent ratoon, and these new varieties were distributed in all sugarcane planting areas[19]. At this stage, excellent germplasm resources bred through nobility process were used as hybrid parent, to further consolidate excellent genes and select better modern sugarcane varieties. (v) The stage of expanding variety genetic constitution source. At this stage, it mainly was engaged in rapidly improving sugar content, yield and other agronomic traits through constant hybridization or backcrossing to increase fundamental substances[20].

3.2BreedingofsugarcanevarietiesinbothChinaandforeigncountriesNew sugarcane varieties bred by foreign countries mainly include RB varieties bred by Brazilian Federal University and IAC varieties bred by Campinas Agricultural Research Institute of Sao Paulo[21], such as RB99395 and IAC86-2210; CP series, H series, and HoCP series bred by USDA ARS Sugarcane Field Station Canal Point, Hawaii Research Institute, and Louisiana Sugarcane Research Institute[22]; Co997, Co1001, and Co527 bred by India[22]; Q174, Q205, and Q208 bred by Australia[23]; POJ and EK varieties bred by Indonesia. The breeding of sugarcane in China can be divided into 3 stages[24-25]. (i) Breeding of local varieties. This stage was marked by planting local varieties as sugar refining materials, typical varieties included bamboo cane, reed cane, and Rohan cane. (ii) Introduction of foreign varieties. At this stage, foreign varieties were introduced and popularized, and typical varieties included POJ2878, POJ2725, NCo310, Co290, Co 281, CP49-50, and CP34-120. (iii) Self-breeding varieties. At this stage, varieties bred by China started replacing foreign varieties. In recent years, self-breeding varieties of varieties mainly included Yuetang, Guitang, Mintang, Yunzhe, Liucheng, and Taitang. At present, new Taitang 22, Yutang 93-159, Yutang 00-236, Guitang 21, and Liucheng 05-136 take up the dominant position of sugarcane planting in China. However, the growth rate for sugar content and yield of new sugarcane varieties becomes smaller and smaller. This can be proved by evaluation of 4 main varieties, Yuetang 85-177, Yuetang 99-66, Yuetang 00-236, and Yuetang 03-393. Waclawovskyelal.[26]found that the growth rate of sugarcane yield in the world remained at 1%-1.5% in recent years, and it will decline in future. This may be largely because breeding parent mainly comes from F4 and F4 of POJ2878[27]and germplasm resources for improvement are very limited. In this situation, biotechnology may become the key for sugarcane genetic improvement.

4 Advances in studies of sugarcane genetic improvement through biotechnology

4.1SugarcanegenomicsSugarcane genomics is an indispensable tool for future sugarcane improvement. However, complex genome of allopolyploid and interspecific hybridization of modern strains hinder studies of sugarcane genomics and application of genomics in sugarcane breeding process. By now, in NCBI database, there are 31555 nucleotide sequences (including 1216 mRNA sequences) available from different sugarcane varieties and 284818 EST sequences (http://www.ncbi.nlm.nih.gov/). These EST sequences come from cDNA library and many transcripts of more than 70 varieties of sugarcane, and materials mainly include seedlings, roots, stalks, leaves, flowers, and seeds of sugarcane, and callus treated by nonbiological stress and seedlings infected by endogenous nitrogen-fixing bacteria[28]. At present, bacterial artificial chromosome (BAC) library for sugarcane is built by hybrid strain R570 with number of chromosome of 2n= 115. This library contains 283158 clones and covers 1.3 times of polyploidy genome of this variety (it is predicted that the genome is 10 Gb)[29]. It is reported that scientists from Brazil and other countries are building fine physical map for this BAC library[30]. Meanwhile, scientists are building BAC library and the library clone sequencing for Brazilian variety SP80-3280[31]. Besides, in cooperation with Shenzhen BGI, Sugarcane Research Center of Chinese Academy of Agricultural Science is building BAC library and whole genome sequencing for thin stalk wild sugarcane variety GXS87-16 (2n=64)[32]. The building of sugarcane BAC library and detailed physical map information are of utmost importance to understanding structure of sugarcane genome. In the whole genome sequencing, sorghum is a crop with close affiliation with sugarcane, and its genome sequence is of much help for studies of sugarcane genome, and completion of sorghum genome sequencing provides important comparative genomics tool for sugarcane genomic studies[33-34]. Wangetal.[35]carried out hybridization in BAC library using 1961 single copy sorghum oligonucleotide probes and sugarcane commercial variety R570, obtained 20 sugarcane BACs, with each BAC corresponding to sorghum chromosome arm. About 95.2% sequences of coding area of sugarcane BACs match the sorghum sequences. If using sorghum genome as template to sequence the contig, it can cover 78.2% of 20 BACs. About 53.1% sugarcane BACs match sorghum sequences. In areas that can be linked, 209 genes of sugarcane have been annotated, 202 sorghum genes have been annotated, including 17 genes unique to sugarcane, and all have been verified by sugarcane expressed sequence tags (ESTs), in 12 genes unique to sorghum, only one has been verified by sorghum ESTs. In 17 genes unique to sugarcane, 12 genes do not have matching protein in GenBank non-redundant protein database, and they may other types of protein participating in coding sugarcane special process. Relative to the sugarcane, lineal homological area of sorghum expands, which is realized mainly through increase in reverse transcription transposon. Therefore, sugarcane and sorghum genomes are collinear in most gene areas. Sorghum genome can be used as DNA assembly template of allopolyploid sugarcane genomes.

4.2SugarcaneTransgenetechnologyThe in vitro culture and regeneration system technology have been established and gradually improved since 40 years ago[36], which is very important for development of sugarcane genetic transformation system. Nevertheless, aneuploidy polyploidy, huge genome and complex genetic background of sugarcane present the problem of low transformation efficiency of transgene technology. Since gene gun method (particle bombardment) features not subject to host, wide target receptor type, high controllability, simple and rapid operation, it is a method mainly used in the early period of the sugarcane transgene technology[37-38]. With constant development and optimization of genetic transformation methods with features of low costs, high success rate, and single copy of allogenic genes, and high genetic stability, it has been widely applied in sugarcane transgene technology[39]. In the process of sugarcane genetic transformation, main restrictive factors include low transformation efficiency, active transgene, mutation in body cell clone, and difficult backcrossing[40]. It is thus required to further optimize the transformation method, better control the transgene expression, and realize stable expression. The sugarcane transgene studies focus on increasing sucrose accumulation, stalk yield, improving disease resistance and stress resistance[40-45]. Researches indicate that genes participating in metabolism of cell wall have differences in expression[40]. Through cDNA-SCoT analysis of ratoon stunting disease induced sugarcane difference expression gene, they found many genes participating in interaction of ratoon stunting disease[46]. Through subtractive library technology and cDNA chip technology analysis, SSADH related to sugarcane water stress response was screened[47]. Through excessive expression or downward modulation of virus coat protein, mRNA exerts resistance against SCMV[48], SCYLV[49], and FDV[50]. Through adjusting expression of Sc-ERS gene, it is able to strenthen photosynthesis of sugarcane leaves and improve drought resistance of sugarcane[51]. These key genes are favorable for cultivation of new sugarcane varieties with high yield, high sugar content, and high adaptation. At present, non-commercial sugarcane transgene strains have made breakthrough advances and some strains are undergoing the field experiment[21,40]. However, limitation of regulations of transgenic crops will retard the release of commercial transgenic varieties. Therefore, the breeding and application of a new transgenic variety takes a considerable long time.

4.3MolecularmarkerassistedbreedingAt present, the number of sugarcane chromosomes for genetic mapping analysis is more than 100, but genome sequence for molecular marker is very limited. Most genetic maps are based on dominant markers, which are used as single dose markers. Posterity segregation ratio exists as per 1:1 (marked as "1"), does not exist (marked as "0") for statistical analysis. For allopolyploid sugarcane, such statistical method will only provide an approximate value when estimating the recombination rate and linkage; besides, some evidences indicate that single dose marker only detects about 70% polymorphism loci[52]. 13 sugarcane mapping groups were used to build 18 molecular genetic linkage map, and 1500-2000 markers were used[53], including RFLP[54], AFLP[55], TRAP[54], EST-SSR[56]and DART[57], indicating all built sugarcane genetic maps are incomplete. To obtain high density genetic maps covering the whole sugarcane genome, it needs developing more SNP markers. However, through QTL positioning of sugarcane related traits, it has obtained QTL loci related to disease resistance, stress resistance, yield, and sugar content[58-62]. Because of complexity of sugarcane genome, for target traits, most genomes can not be scanned, and such defect limits the application of the molecular marker assisted selection. At the same time, the sugarcane genetic mode indicates that the genetic linkage unbalance widely exists[63]. When trait related molecular markers are used to determine QTL of sugarcane through linkage analysis and correlation analysis, low density marker and rough genetic statistical methods are still relatively difficult. In sugarcane breeding, it is a challenging task to use molecular marker assisted selection. Many important traits are jointly determined by many trait loci, and each trait only contributes a little to the overall phenotype[64]. The sugarcane QTL positioning is mainly based on single dose marker analysis or composite interval mapping[63]. To obtain effective results, it needs developing new research models and statistical methods, and also considers influence of interaction between QTL and environment and gene correlation. Therefore, there are still many challenges to be solved in sugarcane molecular marker assisted breeding.

5 Conclusions

Sugarcane is an essential sugar crop and energy crop, and its variety improvement is receiving close attention. Traditional breeding and cultivation techniques have contributed a lot to increasing sugarcane yield and sucrose content. With rapid development of modern biotechnology, relying on its importance in agriculture and industries, sugarcane attracts many scientists to make joint efforts in molecular biology, bioinformatics, and genetics. Besides, with application of new generation of low cost DNA sequencing technology, the allopolyploid sugarcane genome sequencing which was costly in the past becomes possible. In future, biotechnology genetic improvement technique will accelerate the progress of traditional sugarcane breeding and cultivate more excellent sugarcane varieties.

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