Feasibility of DNA Barcoding for Definition of Forage Mulberry

Chan ZHOU, Jieping WANG, Yao ZENG, Shanlin GU, Jinfeng LU

Institute for Silk and Related Biomaterials Research, Chongqing Academy of Animal Sciences, Chongqing 402460, China

Abstract The planting and afforestation of mulberry and its application as animal feed conform to the spirit that comprehensive utilization of biomass such as agriculture and forestry is the direction of sustainable development in the National Program for Medium-to-Long-Term Scientific and Technological Development. The application of mulberry as animal feed has attracted international attention, but the cultivar identification of forage mulberry is still controversial in the industry. The paper summarizes the current situation of forage mulberry in animal husbandry, and expounds how to define and classify forage mulberry.

Key words Mulberry; Feed; Definition; Animal husbandry

1 Introduction

Mulberry planting and silkworm rearing is a traditional agricultural industry in China. Mulberry is a kind of fine eco-economic forest with high nutritional value, high yield, strong adaptability and easy cultivation. Mulberry has been planted in China for more than 4 000 years.Morusplants are fast-growing woody plants with deep roots and luxuriating leaves, pollution resistance, wind and sand resistance, drought resistance, salt and alkali resistance, strong adaptability, easy cultivation, air purification effect, wide range of available resources, and great market potential, which are eco-economic trees with huge economic, ecological and social value[1-3]. Traditional mulberry planting is mainly used for silkworm rearing, so a large number of mulberry leaves become a wasted resource due to surplus. The total area of mulberry gardens in China is 7 986.5 hm2. With the decline of cocoon and silk industry, the utilization rate of mulberry gardens is less than 50%, and a large number of mulberry gardens are idle, resulting in a great waste of resources. With the promotion of the projects of "moving mulberry from east to west" and "returning farmland to forest", there are about 800.4 hm2of mulberry gardens in Chongqing, with the cocoon yield of about 20 000 t. The benefit of mulberry industry is extremely low, and the economic value of mulberry has not been fully developed.

With the continuous growth of livestock and poultry industry in China, animal husbandry consumes about 200 million t of feed grain every year, while the feed grain consumption increases at an annual rate of about 1.5%-2.0%, and the annual feed grain consumption is increased by about 3 million to 4 million t. Animal husbandry is facing with a prominent problem of feed grain supply and demand difficulties. Studies have shown that the crude protein content in the leaves of someMorusplants is up to 22%-25%. Bai Xuhua[4]pointed out that the amino acid composition of protein in mulberry leaves was similar to that of defatted soybean powder. According to a report by the UN Food and Agriculture Organization (FAO) and the Livestock Production Department of Ministry of Agriculture (AGAP), mulberry leaves produce more nutrients than most traditional feeds from the perspective of digestible nutrition, and can be used as a source of vegetable protein feed for livestock and poultry, to alleviate feed shortage in animal husbandry. Mulberry leaves, as livestock and poultry feed resources, have a huge market prospect. The most remarkable characteristic of mulberry leaves as animal feed, especially green feed, is its high digestibility.Invivoandinvitrotests have proved that the digestibility of mulberry leaves in animals is 70%-90% under normal conditions. Another important property of forage mulberry leaves is that they have good palatability to all livestock and are easily accepted by the animals who first come into contact with mulberry leaves without feeding disorders. As a supplement for young calves, mulberry leaves can save the consumption of milk or milk replacer and promote rumen development and growth of calves. Recently, there have been reports of using mulberry leaves to raise rabbit, deer and fish.

At present, a large amount of fecal sewage is produced in large-scale livestock and poultry breeding, which causes great pollution to the ecological environment and even endangers food safety and human health.Morusplants have well-developed roots and prefer deep, moist and fertile soil, and require a large amount of organic fertilizers in the growth process. The concept of ecological cycle is adopted to effectively combine livestock and poultry fecal disposal with mulberry cultivation and management, and the measures such as fermenting pig feces into green energy biogas through biogas fermentation technology, developing biogas residue into organic fertilizer, and using biogas slurry for mulberry irrigation not only reduce the pollution of feces to the environment, but also provide environmentally friendly clean fertilizer and energy forMorusplants to help their rapid growth and increase economic benefits. The measures solve the problem of fecal disposal in large-scale livestock and poultry breeding while ensuring the growth nutrition of forageMorusplants.

2 There is no strong basis for the taxonomy of forage Morus plants

In 1937, Chen Rong, a botanist from China, divided the genusMorusin China into 5 species and 7 variants in theTaxonomyofChineseTrees. Afterwards, Hu Xiansu divided the genusMorusin China into 8 species. According to the literature records of plant taxonomists at home and abroad onMorusspecies, various provinces and regions have carried out investigations on mulberry germplasm resources and collected a large number of material objects. After sorting and identification, there are 15 species and 4 variants in China which is the country with the largest distribution ofMorusspecies in the world. The lack of a complete taxonomy system forMorusplants is partly due to the fact that mulberry is an alien plant, which is wind-pollinated with many opportunities for natural hybridization. Moreover, frequent spontaneous mutations such as bud mutation and branch mutation increase the genetic variability of mulberry. The genusMorusin China is composed of cultivated species and wild species. The cultivated species includeM.alba,M.albavar.multicaulis(Perrott.) Loud.,M.atropurpureaandM.mizuhoHotta.; the wild species includeM.wittiorumHand.-Mazz.,M.macroura‘Long Fruit’, L.,M.cathayanaHemsl.,M.serrataRoxb,,M.mongolicaSchneid. var.diabolicaKoidz,M.mongolica(Bur.) Schneid.,M.notabilisSchneid.,M.nigriformisKoidz,M.australisPoir.,etc.; the variants includeM.mongolicavar.diabolicaKoidz.,M.alba‘Pendula’,M.albavar.venoseDelile.,etc., distributed in different areas of China[5-6]. Experts of Beijing Sangliang Technology Development Center cultivated Yajin protein mulberry through artificial selection and cross breeding, called forage mulberry. However, the biological classification of forage mulberry has not reached a definitive conclusion in the academic circle.

3 DNA barcoding is expected to give biological definition of forage Morus plants

Forage mulberry is a new term which appears with the research and application of mulberry in animal husbandry in recent years. However, the definition of forage mulberry has been controversial in academic circles. Variety selection, planting environment, annual leaf yield and protein content are indicators of forage mulberry that should be cleared. Our research team has applied forage mulberry in large-scale pig breeding, and achieved certain results. ForageMorusplants for livestock and poultry breeding should have the following features: (i) mulberry plants should be harvested 6-8 times a year, and leaves have very low lignification with a protein content of 30%; (ii) there is no lignified fiber within 70 cm of mulberry branch, and at least 28% plant protein; (iii) mulberry plants have wide planting adaptability and strong stress resistance, and can be planted in a large range; (iv) mulberry plants have strong ability to absorb feces, and can be planted around large-scale farms.

With the development of studies on sexual cross-breeding, mutagenesis breeding and polyploid breeding of mulberry, it is limited to distinguish forage mulberry cultivars solely from epigenetic characteristics[7-8].

DNA barcoding is a fast and accurate technique for species identification. The technique uses relatively universal, short DNA fragments with sufficient variation and easy amplification to create a new biological identification system, so as to rapidly identify species. It has been a hot spot in the international genetic taxonomy and identification research in recent years, and has shown broad application prospects in the field of species identification. DNA barcoding is expected to define forageMorusplants from the perspective of molecular identification[7-10].

4 DNA barcoding is a stage in the development of biotaxonomy

DNA barcoding is a fast and accurate technology for species identification. By comparing the DNA sequence of a certain gene with the standard sequence in the database, rapid identification of species can be realized. It has been widely used in the fields of ecology, environment, food safety, customs quarantine, disease prevention and control, and biodiversity protection[11-14].

The real concept of DNA barcoding was proposed by Hebert and other scholars in 2003. Hebertetal.[15]held that DNA barcoding is a technology that uses one or a few DNA fragments as standard fragments for rapid, accurate, and automated labeling of existing species on the earth. The application of DNA barcoding in the identification of animal species was also started by Hebert in 2004. He analyzed the cytochromic C oxidase I gene sequences of all animal phyla except Cnidaria (11 phyla and 13 320 species in total) in Animalia, and found that the differences in sequences could well distinguish these species[16-17]. Therefore, Hebert and other scholars insisted that cytochrome C oxidase I(COI) is the most suitable DNA barcoding standard gene in Animalia[15-18].

DNA barcoding has been relatively mature in animal taxonomy, and the generally accepted good DNA barcoding standard fragments have been revealed. However, the researches in plant classification did not go so well. Because mitochondrial genes of plants have slow evolution rate, with small gene fragment variation and low genetic differentiation,COIis not suitable to be used as a barcoding fragment. Therefore, the research of DNA barcoding in plant taxonomy progresses slowly[10-15]. Chaseetal.[19]usedrbcLgene andITSgene, the molecular marker genes commonly used in the phylogeny of terrestrial plants, for species identification and analysis, and finally the sequence variation of the genes obtained could clearly identify the species. Therefore, Chase put forward that it is possible to find a uniform standard DNA barcode for reliable species identification of terrestrial plants.

ITSsequence is a fragment of ribosome gene in the nuclear genome, which is widely found in terrestrial plants and is the most commonly used fragment for systematic studies. Due to the development of researches on barcoding, large amounts ofITSdata have been stored in GenBank.ITSsequence contains two transcriptional spacers, namelyITS1 andITS2, andITS2 is better thanITS1 in species identification.ITS2 sequence is short, with a total length of about 200 bp, rapid evolution rate and high variability. All these characteristics enableITS2 sequence to have better species identification ability. In terms of single gene identification, the identification ability ofITS2 in all species is up to 54.6%. However, such recognition ablity is still very low and can not meet the requirements of barcodes[18,20]. Therefore, when screening plant barcoding, many scholars recommend the method of combing multiple fragments.rbcLandmatKsequences in the chloroplast genome have also been proposed as DNA barcodes for plant identification.

rbcLsequence in the chloroplast genome is a very important gene affecting chloroplast photosynthesis. The protein encoded byrbcLis the large subunit of ribulose-1,5-bisphosphate carboxylase. Due to the conserved position and slow mutation rate,rbcLgene has been studied as plant DNA barcode by more and more scholars. However, the overall length ofrbcLgene sequence is too long (about 1 300 bp) to be fully and accurately amplified and sequenced by a single primer. Therefore, Kressetal.[21]suggested that part of these segments could be selected as the barcode for amplification, and the selected segment was namedrbcL-a.

marKsequence is in the intron of lysine gene (trnK) in the chloroplast genome, encoding a mature enzyme K and involving in type II intron splicing in the RNA transcriptome[22-23].matKsequence is a single copy gene with a total length of about 1 500 bp. It is a protein-encoding gene in the chloroplast genome that evolves quickly. Gao Tingetal.[24]also preliminarily discussed the application ofmarKgene in the identification of legumes, and concluded that the identification ability ofmatKin legumes was 73%-80%. The full length ofmatKgene was also too long, and a part of about 850 bp was taken for amplification.

Finally, the plant working group of the consortium for the barcode of life (CBOL) decided to use the two gene fragments,rbcLandmatK, in chloroplast as the nuclear barcodes of plant DNA standard barcodes, and theITSfragment in nuclear genes andtrnH-psbAfragment in chloroplast as the supplementary barcodes of plant DNA barcodes[25].

Due to a wider diversity of plants and the technical limitations of DNA barcoding, no recognized universal DNA barcoding sequence has been found in plants, butITS2,psbA-trnH,rbcL,matKand other sequences are considered to be the most potential and suitable DNA barcoding sequences for plants. DNA barcoding identification can only guarantee the reliability of results if samples are accurate and distribution area includes all the variations based on morphological classification studies. Therefore, DNA barcoding identification and traditional morphological taxonomic studies are complementary and need to be closely integrated[20,22-26].

5 Prospect

China has rich mulberry germplasm resources. The development and utilization of mulberry in animal husbandry with its rich nutrition and high protein content can not only improve the economic benefits of sericulture, but also solve the pollution of by-products of animal husbandry, which is conducive to environmental protection and conforms to the direction of sustainable development. The development and utilization of forage mulberry is still in the exploratory stage, and needs to carry out systematic research and mature mode exploration. Therefore, we should attach importance to the development of basic research, establish a set of standards for evaluating the nutritional value of mulberry, and establish a scientific and technological innovation system for comprehensive utilization of mulberry resources in animal husbandry. The development of forage mulberry should implement the integration, regionalization and specialty relying on industrialized bases, and develop circular economy by learning from other mature models.