Innovation on Distant Hybridization of Saline-tolerant Mud Flat Spartina and Rice Germplasm

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1. Nantong City Kangbole Agricultural Science and Technology Co., Ltd., Nantong 226001, China; 2. Nanjing Haipeite Agricultural Science and Technology Co., Ltd., Nanjing 210007, China

1 Introduction

There are 15 varieties ofSpartinain the world, all of them are perennial C4 herbs and have certain saline and flood tolerance[1].Spartinaanglica(cordgrass) andSpartinaalterniflora(smooth cordgrass) (hereafter referred to asSpartina) are varieties with the largest area of distribution in coastal regions of China.Spartinaanglicawas introduced by Nanjing University from Britain in 1963, and the number of chromosomes is 122 and 124.Spartinaanglicahas been included into the Feed Science of China, has strong saline and flood tolerance, and is suitable for mid tide zone. The annual yield of freshSpartinaanglicais 15-30 t/hm2, suitable for feeding ruminants[2].Spartinaalterniflorawas introduced by Nanjing University from the United States of America in 1979, and it has 62 chromosomes.Spartinaalternifloraalso has strong saline and flood tolerance, but the plant is higher thanSpartinaanglicaand the grass yield is high, suitable for feeding herbivores, and thus receives wide attention[3].

Graduated from Nanjing Agricultural University in 1975, Chen Qikang has been studyingSpartina. Under the guidance of Professor Zhong Chongxin from Nanjing University, he introducedSpartinaanglicain 1975 andSpartinaalterniflorain 1980, and built 80000 mu tidal flatSpartinapasture, and established the firstSpartinapasture of China. Besides, he is undertaking the Program of Coastal Pasture Experiment of State Science and Technology Commission, and the coastal pasture experiment was identified as the first in China; theSpartinacan feed 3200 cattle and sheep every year[4-6]. With more than 40 years of study onSpartinaas feed of livestock and poultry, Chen Qikang found thatSpartinahas excellent properties of saline and flood tolerance, but the yield is low and insufficient to satisfy nutritional demand of cattle and sheep, so he was resolved to explore saline tolerant gene resources ofSpartina. Rice belongs to graminaceous C3 plant with 2n=24 chromosomes. Rice varieties are abundant, seeds are plump, the yield is high, and rice chaff and straws can be used as feed for cattle and sheep. According toFeedScienceofChinacompiled by Zhang Ziyi, rice contains 8% crude protein, nitrogen-free extract (NFE) content ranks the first in various grains, and the available energy is also the highest, so it is an excellent energy feed[2]. Rice provides excellent germplasm resources forSpartinamodification. Distant hybridization of rice can introduce favorable genes of paternal plants, generate non-comparable diversity and new varieties, and expand genetic basis of rice[7]. To explore saline tolerant and high yield germplasm resources ofSpartinaand Rice, we carried out distant hybridization study onSpartinaand Rice in 2005-2016.

2 Materials and methods

2.1Parentalmaterialsfordistanthybridization

2.1.1Mud flatSpartina. (i) In 2005-2006, we introduced and cultivated 1720 plants ofSpartinaanglicaand 560 plants ofSpartinaalterniflora. The plants ofSpartinaanglicawere purchased from Jiangsu Qidong Huanghai Sea Beach Development Co., Ltd (former Qidong mud flatSpartinapasture); (ii) in 2014-2016, we introduced and cultivated 680 plants ofSpartinaalterniflorafrom Tongzhou Bay in Nantong of Jiangsu Province by three times. We took them as parental materials of rice hybridization.

2.1.2Rice varieties. In 2005-2016, we introduced and cultivated following rice varieties as parental materials of rice hybridization: 1. Dahua Xiangnuo; 2. Jindao 1007; 3. Wuxiangnuo 8333; 4. Huajing No. 4; 5. Yinxiang 18; 6. Yanjing No. 10; 7. Wuyunjing No. 7; 8. Wujing 15; 9. Chengnuo 218; 10. Zhenxian 866; 11. Ningjing No. 1; 12. Tongyu Jing No. 14; 13. Nanjing 45; 15. Nanjing 47; 16. Nanjing 5055; 17. Shangshi 400; 18. Nipponbare; 19. 7K339; 20. Wujing 13; 21. Yandao No. 9; 22. Nongken 57; 23. Youfujing; 24. Yanjing 98; 25. Yanfeng 47; 26. Zhonghua No. 1; 27. Zhongxiang No. 1; 28. Liuqianxin; 29. Changnongjing No.4; 30. Yangdao No. 6.Spartinablooms in the end of July, and becomes exuberant in August to October. To make the florescence coincide with rice, we sowed one batch (4 batches in total) every 15 days from April 20.

2.2DistanthybridizationmethodofSpartinaandRiceIn 2005-2007, we undertook Rice ♀×Spartina♂ hybridization experiment. We did warm water lanching and ale gametocide treatment of Rice ♀, cut stamen ofSpartinato pollinate, to explore whether hybridization of Rice andSpartina. In 2008-2013, on the basis of successful experiment of Rice ♀×Spartina♂ hybridization, we expanded the scope and quantity of hybridization. In 2014-2016, we undertook the study on reciprocal distant hybridization ofSpartinaand Rice. Normal hybridization: Rice ♀ warm water lanching and ale gametocide treatment, cut stamen ofSpartinato pollinate, and did steam lanching and ale gametocide treatment to pollinate; reverse hybridization: continuously cut stamen to pollinate forSpartina♀.

2.3Rice♀×Spartina♂distanthybridizationmethodStep 1: takingSpartinaas male parent and rice as female parent, we carried out genus distant hybridization, obtained the first generation of hybrid, conducted screening test of planting in saline-alkaline land, phenotypic screening, molecular marker assisted selection, and finally obtainedSpartina/Oryzasativahybrid seeds containingSpartinaand Rice. Step 2: using backcross method + molecular marker assisted selection method, we selected new varieties (lines) ofSpartina/Oryza sativa suitable for growth in mud flat saline-alkaline land. We made a record of crossbreeding, backcross, and molecular marker assisted selection, and annually prepared file of hybrid pedigree, and kept samples of crossbreeding and backcross seeds in seed bank for reference.

2.4Studymethods

2.4.1Patent for invention. Genus distant hybridization methods ofSpartinaand Rice on mud flat[8]. Patent number: ZL 2007 1 0019635.6.

2.4.2Three-in-one technology. We combined mud flat planting screening, phenotypic screening, and molecular marker assisted selection methods to breakthrough key technology of saline tolerance biological characteristics.

2.5RAPDmolecularidentificationofmudflatSpartina/RicedistanthybridizationmaterialsRAPD molecular identification of mud flatSpartina/ Rice distant hybridization materials was carried out by Professor Qin Pei from Institute of Biological Technology of Nanjing University, Associate Professor Tian Zengyuan from School of Life Sciences, Zhengzhou University, and Associate Professor Chen Shihua from College of Life Sciences of Yantai University. Detection samples: we used D to denoteSpartinaanglica, H to denoteSpartinaalterniflora, R to stand for rice, and RD1-n and RH1-n for hybrid seeds.

3 Results and analyses

3.1ExperimentofSpartinaandRicehybridizationandmudflatplantingin2005-2008In 2005, we obtained 149 seeds from hybridization of Peiai 64S/04141(7K339♀) R and D (♂). In 2006, we obtained 224 ears of F1generation selfed seeds, 11842 grains of selfed seeds, 1000-grain weight 18.38 g; F1(♀)×D(♂) backcross 17 ears, obtained 24 grains of BC1F1seeds, the seed setting rate of backcross 2.84%; 6 varieties of R(♀)×D(♂) hybrid 23 ears, 63 grains of seeds, and the seed setting rate of crossbreeding 4.2%[9,10]. We introduced and cultivated 560 plants ofSpartinaalterniflorain 2005, and carried outSpartinaalternifloraand Rice hybridization experiment in 2006. Results indicate that R(♀) 7 varieties (groups) × H (♂) hybridization 39 ears obtained 115 grains of hybrid seeds, with seed setting rate of 5.4%; F132, F134(♀) × H (♂) hybridization 12 ears obtained 18 grains of hybrid seeds, with seed setting rate of 2%. In December 2006, the Institute of Biological Technology of Nanjing University measured samples of hybrid seeds,Spartinaanglicaand Rice hybrid varieties: RDF132 and RDF134;Spartinaalternifloraand Rice hybrid varieties: RHF137 and RHF139, RAPD reaction indicates that compared with rice parental materials, the strap of hybrid generation had distinct difference, indicating that distant hybridization leads to recombination of rice genes[11,12].

To increase quantity of new germplasm and expand scope of germplasm resources ofSpartinaand Rice hybridization, we hybridized R(♀)×D(♂) (RD) for 110 groups in 2007, the seed setting rate up to 20.3%, we hybridized R(♀)×H(♂) (RH) for 50 groups in 2007, the seed setting rate up to 23.1%. In the same year, we rented 1.33 hm2mud flat in Xingken of Qidong City and carried out saline tolerance adaptation experiment forSpartinaand Rice hybrid seeds. Under the conditions of soil salt content 7.83‰, pH 8.20, and irrigated water 10.37‰, 1000-grain weight ofSpartinaand Rice hybrid F2 was 18g, seed setting rate was 52.8%, and single plant seed weight was 19.97 g. In 2007, under the conditions of soil salt content 5.28‰ and pH 7.9, 1000-grain weight ofSpartinaand Rice hybrid F2 reached 22g, seed setting rate reached 51.1%, and single plant seed weight was 19.1g. 1000-grain weight of rice × maize ×Spartinahybrid seeds was 22.8g, seed setting rate reached 69.9%, and single plant seed weight up to 41.4g; in experiment of Institute of Agricultural Sciences of the Area along Yangtze of Jiangsu Province, 1000-grain weight ofSpartinaanglicaand Rice F2 hybrid seeds reached 22.4g, seed setting rate was 58.2%, and single plant seed weight was 33.7%[13,14]. In 2008, saline tolerance adaptation experiment was carried out forSpartinaanglica/ Rice hybrid seed No. 34 and No. 32, and 3.72 t/hm2hybrid seeds were obtained. In 2005-2008, we hybridized Rice (♀) ×Spartina(♂) 358 groups.

3.2InnovationonSpartinaandRicehybridizationandnewsalinetolerantSpartina/Ricehybridvarietiesin2009-2012Based on successfulSpartinaand Rice distant hybridization and trial planting in mud flat saline-alkaline land in 2005-2008, we carried out innovation onSpartinaand Rice hybridization and new saline tolerantSpartina/ Rice hybrid varieties in 2009-2012. We hybridized Rice (♀) ×Spartina(♂) 669 groups in 2009-2012. Hybridization results: 421 groups of R(♀) × D(♂) (RD) were hybridized, and 248 groups of R(♀) × H(♂) (RH) were hybridized. A total of 289 samples ofSpartina/Rice hybrid seeds were detected in 2009-2012. Among them, 48 samples had genetic elements of parentalSpartinaandOryzasativa, and we screened new salineSpartinaandOryzasativahybrid varieties. In 2009, through mud flat screening combined with molecular marker assisted selection, in 32 groups, 20 groups obtained saline tolerant rice higher than 3.0 t/hm2with the average rice yield 4.89 t/hm2(the minimum yield was 3.11 t/hm2and the maximum yield was 8.94 t/hm2). We selected 6 new saline-tolerantSpartina/Rice hybrid varieties with yield of 6.0-7.5 t/hm2. We found breeding materials of saline tolerant sweet stalk japonica rice germplasm resources, soluble sugar content of straw was above 5.0%, which was more than 65% higher than the control group. In 60 samples of hybrid seeds, we selected 18 hybrid groups. Through RAPD analysis, 9 groups had common strap with parentalSpartinaanglicaand had vacancy of parental Rice materials, indicating that 9 groups of distant hybrid seeds had genetic elements of parentalSpartinaanglica[15,16].

3.2.1Results ofOryzasativa♀ ×Spartinaanglica♂ hybridization. In 2009-2011, we obtained experimental evidence of seed setting and transplantation ofSpartinaanglicaand Rice distant hybridization, and economic traits of hybrid materials; through RAPD analysis, 22 samples of hybrid materials had common straps with parentalSpartinaanglicabut no parental Rice, indicating that 22 samples of distant hybridization materials had genetic elements of parentalSpartinaanglica[17]. In 2009, we hybridized 30 groups of seeds, cut hull of 1737 grains, obtained 409 grains of seeds, and seed setting rate reached 22.1%. In 2010, we sowed 30 groups of hybrid seeds, transplanted 8 groups, the germination rate reached 26.67%; we sowed 409 grains of seeds, transplanted 18 grains, and the germination and transplantation rate was 4.4%. Analysis: in 2009, we cut hull of 1737 grains; in 2010, we transplanted 18 grains, the actual hybridization success rate was 1.04%. In 2010, we hybridized 3 groups of seeds, cut hull of 126 grains, obtained 31 grains of seeds, and seed setting rate reached 27.1%. In 2011, we sowed 3 groups of hybrid seeds, one group germinated, the germination rate reached 33.33%; we sowed 31 grains of seeds, transplanted one grain, and the germination and transplantation rate was 3.2%. Analysis: In 2010, we cut hull of 126 grains; in 2011, we transplanted one grain, the actual hybridization success rate was 0.79%. Experimental results of these two years: success rate of Rice ♀×Spartinaanglica♂ hybridization was 0.79-1.04%.

3.2.2Results ofSpartinaOryzasativa♀ ×Spartinaanglica♂ backcross. In 2009, we hybridized 11 groups of seeds, cut hull of 769 grains, obtained 176 grains of seeds, and seed setting rate reached 21.64%; in 2010, we sowed 11 groups of hybrid seeds, transplanted 7 groups, the germination rate reached 63.64%; we sowed 176 grains of seeds, transplanted 15 grains, and the germination and transplantation rate was 8.52%.Analysis: In 2009, we cut hull of 769 grains; in 2011, we transplanted 15 grain, the actual hybridization success rate was 1.95%. In 2010, we hybridized 13 groups of seeds, cut hull of 679 grains, obtained 75 grains of seeds, and seed setting rate reached 9.52%. In 2011, we sowed 13 groups of hybrid seeds, transplanted 4 groups, the germination rate reached 30.77%; we sowed 75 grains of seeds, transplanted 12 grains, and the germination and transplantation rate was 16%. Analysis: in 2010, we cut hull of 679 grains; in 2011, we transplanted 12 grains, the actual hybridization success rate was 1.77%. Experimental results of these two years: success rate ofSpartinaOryzasativa♀ ×Spartinaanglica♂ backcross was 1.77-1.95%.

3.2.3Results ofSpartinaOryzasativa♀ ×Oryzasativa7K339♂ backcross. In 2009, we hybridized 130 groups of seeds, cut hull of 8630 grains, obtained 2509 grains of seeds, and seed setting rate reached 26.89%. In 2010, we sowed 130 groups of hybrid seeds, 45 group germinated, the germination rate reached 34.62%; we sowed 2509 grains of seeds, transplanted 177 grain, and the germination and transplantation rate was 7.05%. Analysis: In 2009, we cut hull of 8630 grains; in 2010, we transplanted 177 grain, the actual hybridization success rate was 2.05%. In 2010, we hybridized 43 groups of seeds, cut hull of 2600 grains, obtained 1134 grains of seeds, and seed setting rate reached 35.27%. In 2011, we sowed 43 groups of hybrid seeds, 16 group germinated, the germination rate reached 37.21%; we sowed 1134 grains of seeds, transplanted 49 grain, and the germination and transplantation rate was 4.32%. Analysis: In 2010, we cut hull of 2600 grains; in 2011, we transplanted 49 grain, the actual hybridization success rate was 1.88%. Experimental results of these two years: success rate ofSpartinaOryzasativa♀ ×Spartinaanglica7K339♂ backcross was 1.88-2.05%.

3.2.4Results of Rice ♀ ×Spartinaalterniflora♂ hybridization. In 2009-2011, we undertook study ofOryzasativaandSpartinaalternifloradistant hybridization, appliedOryzasativaandSpartinaalternifloragenus distant hybridization methods, and combined mud flat planting screening, backcross phenotypic screening, cell-based screening, and molecular marker assisted selection technologies, to find new saline tolerant rice varieties suitable for planting in mud flat and using as feed. In 2009-2011, the success rate of actual distant hybridization was 1.39%. We randomly selected 6 samples of 7C14 and Zhongxiang No.1 rice female parent andSpartinaalterniflora(H) male parent distant hybrid seeds to make RAPD molecular analysis, found that 10K205-7C14×H, 8K157-7C14×H, 9H5 Zhongxiang No.1 ×H had common strap with parentalSpartinaalterniflora, but no parental Rice, showing that the above distant hybrid seeds had genetic elements of parentalSpartinaalterniflora. Besides, we selected 12 samples of 7K339 rice female parent andSpartinaalterniflora(H) male parent distant hybrid seeds and carried out RAPD analysis, results indicated that 10K215, 8K48, 9H9, 9H8, and 9H28 had common strap with parentalSpartinaalterniflorabut no parental rice, showing that the above distant hybrid seeds had genetic elements of parentalSpartinaalterniflora, while there was different degree of variation in other hybrid seeds, parentalSpartinaalterniflora, and parental rice[18, 19].

3.3BreedingofnewsalinetolerantSpartina/Ricehybridvarietiesin2013-2016

3.3.1Study on innovation methods for reciprocal distant hybridization ofSpartinaalternifloraand Rice. In 2014-2016, we undertook the study on suitable methods for reciprocal distant hybridization ofSpartinaand Rice. Normal hybridization: Rice ♀ warm water lanching and ale gametocide treatment, cut stamen ofSpartinato pollinate, and did steam lanching and ale gametocide treatment to pollinate; reverse hybridization: cut stamen ofSpartinaalterniflora♀ to cut hull to pollinate, and warm water lanching and ale gametocide treatment. Results indicate that Rice ♀ hot steam ale gametocide treatment pollination method could increase the seed setting rate by 34.09%, increase the average ear seed setting by 121.21%, the germination rate by 60.07%, work efficiency by 6-7 times;Spartinaalterniflora♀ continuous ale gametocide treatment pollination could increase the seed setting rate by 3.14 times, the average ear seed setting by 4.21 times, germination rate by 68.74%, and work efficiency by 7-8 times. Practice proved that Rice ♀ hot steam ale gametocide treatment pollination method is suitable for Rice ♀ ×Spartinaalterniflora♂, whileSpartinaalterniflora♀ continuous ale gametocide treatment pollination method is suitable forSpartinaalterniflora♀ × Rice♂[20].

3.3.2Study on innovation methods for reciprocal distant hybridization ofSpartinaanglicaand Rice. In 2014-2016, we undertook the study on suitable methods for reciprocal distant hybridization ofSpartinaanglicaand Rice. The study method is the same as that for reciprocal distant hybridization ofSpartinaalternifloraand Rice. Results indicate that Rice ♀ hot steam ale gametocide treatment pollination method could increase the seed setting rate by 103.23%, increase the average ear seed setting by 65.58%, the germination rate by 88.67%, work efficiency by 5-6 times;Spartinaanglica♀ continuous ale gametocide treatment pollination could increase the seed setting rate by 1.67 times, the average ear seed setting by 2.83 times, germination rate by 41.79%, and work efficiency by 6-7 times. Practice proved that Rice ♀ hot steam ale gametocide treatment pollination method is suitable for Rice ♀ ×Spartinaanglica♂, whileSpartinaanglica♀ continuous ale gametocide treatment pollination method is suitable forSpartinaanglica♀ ×Rice♂. In 2013-2016, we hybridized 471 groups ofSpartinaand Rice.

3.3.3Study on high yield cultivation technologies for mud flat saline tolerantSpartinaRice. In view of characteristics of saline-alkaline mud flat, through implementing measures such as water and soil resource utilization, farming modification, supporting river system construction, seed selection and treatment, water direct sowing, raising rice seedlings on seedbed, field transplantation, weeding, fertilizer and water management, prevention and control of plant diseases and insect pests, the yield of saline tolerantSpartinaRice can reach 5.925-8.28 t/hm2. Then, we summarized high yield cultivation technologies for mud flat saline tolerantSpartinaRice, formulated technical procedures, to provide basis for scientific cultivation of mud flat saline tolerantSpartinaRice[21,22].

3.3.4Breeding of new saline tolerantSpartinaand Rice lines in 2013-2016. On the basis of innovation ofSpartinaand Rice germplasm in 2005-2013, we carried out saline tolerant germination experiment for 223 groups ofSpartinaand Rice hybrid seeds. Under the conditions of the salt content of 15‰ and pH 7.0 water environment, 81 groups had germination rate above 50%, accounting for 36.32%; at salt content of 20‰ and pH 7.0 water environment, 17 groups had germination rate higher than 50%, accounting for 7.62%. In 2014-2016, we selected 20 groups of excellent hybrid seeds, and combined mud flat planting screening, phenotypic screening, and molecular marker assisted selection methods to breakthrough key technology of saline tolerance biological characteristics. Using hybrid breeding, conventional breeding, and molecular breeding methods, we cultivated 10 new lines of saline tolerantSpartinaand Rice suitable for planting in mud flat, and the rice yield reached 5.925-8.28 t/hm2. Among them, 2 lines had yield up to 7.68-8.28 t/hm2. In addition, we established 2 hm2new line cultivation and seed breeding base, made a trial planting of new lines of saline tolerantSpartinaand Rice in 20 hm2mud flat, carried out cultivation demonstration for new line high yield varieties, and formulated technical procedure for high yield cultivation of saline tolerantSpartinaand Rice.

4 Conclusions and discussions

4.1ConclusionsUsing autonomous patent for invention, we studied distant hybridization ofSpartinaand Rice. We adopted tidal flat planting screening, phenotypic screening, molecular marker assisted selection, to find biological evidences of new germplasm, and also applied crossbreeding, conventional breeding, and molecular breeding, to select new saline-tolerantSpartinaand Rice varieties. With 12 years of efforts, we developed 1498 crossbreeding combinations and established pedigree file and seed bank, tested 307 crossbreeding materials (57 hybrid seeds showed parental genetic components ofSpartinaand Rice); obtainedSpartina/Rice distant hybridization and seed setting, transplanting, hybridization material economic traits, and molecular biological experimental evidences; Rice ♀ ×Spartina♂ hybridization success rate of 1.04-1.39%; obtained authorization of patent for invention ofDistantHybridizationMethodsforSpartinaand Rice; formulated procedures for high yield cultivation ofSpartina-Rice, planted 33.33 hm2Spartina-Rice in coastal saline and alkaline land, cultivated 10 new saline-tolerantSpartina-Rice varieties, and the rice yield up to 5.925-8.28 t/hm2.Spartina-Rice is saline-tolerant high yield rice germplasm, developed through sexual hybridization, conventional breeding, and molecular breeding technologies, and is optimal crop for saline and alkaline land. Success ofSpartina-Rice solves the problem of saline-tolerant crop badly for transformation of saline and alkaline land, the problem of feeds for herbivores, as well as problem of environmental pollution resulted from straw burning. Besides, it is of essential scientific significance for resource utilization, increase of agricultural efficiency, grain security, and farmland strategy, so it has broad application prospect.

4.2DiscussionsAccording to statistics of the United Nations Educational, Scientific and Cultural Organization (UNESCO), the area of saline-alkaline land in the world is up to 950 million hm2; in China, the saline-alkaline land is up to 37 million hm2, accounting for 1/28 of the world saline-alkaline land, equivalent to 1/4 existing farmland of China. Using traditional and modern genetic engineering method to raise saline tolerance and cultivate saline-tolerant varieties is a most effective approach for development and use of saline-alkaline land[23]. Distant hybridization, as a breeding means, is mainly applied for introducing useful genes of different genus and varieties, to improve existing varieties. Distant hybrid rice, specially hybrid rice with genes of different genus, may have powerful advantages beyond our imagination[24]. Under the conditions of constant increase in population and grain demand and gradual decline in farmland area and fresh water resources, coastal development and saline-alkaline land transformation projects badly need solving problems such as saline tolerance of crops and high efficient use of straws. Exploration and innovation of favorable genes of distant varieties of crops will become an essential approach for modification of crop varieties.Spartina-Rice is saline-tolerant high yield rice germplasm, developed through sexual hybridization, conventional breeding, and molecular breeding technologies, and is optimal crop for saline and alkaline land. Success ofSpartina-Rice solves the problem of saline-tolerant crop badly for transformation of saline and alkaline land, the problem of feeds for herbivores, as well as problem of environmental pollution resulted from straw burning. Besides, it is of essential scientific significance for resource utilization, increase of agricultural efficiency, grain security, and farmland strategy, so it has broad application prospect.

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