ASSESSMENT OF RICE SORTS AND HYBRIDS ON THE SALT TOLERANCE USING LABORATORY METHODS

The article presents the results of the analysis of salt tolerance in rice samples by the laboratory methods at a concentration of NaCl – 1,5 %. The plants were grown in an incubator at temperature 26 °C. After 15 days, we measured the length of roots and shoots; the germination of seeds was counted. For the analysis of hybrids sensitive to salt Krasnodar varieties Novator with lines IR 52713-2B-8-2B-1-2, IR 74099-3R-3-3, NSIC Rc 106 – donor locus salt tolerance Saltol, using markers RM 493 and RM 7075. 90 plants were selected from the F2 plants which are analyzed by PCR, and the results of DNA analysis identified 6 of them as salt tolerant samples homozygous for the desired locus. From their offspring was selected the promising form to create salt-tolerant rice sorts.


Introduction
Rice is the most important food crop for more than half of the world population.Million of hectares in the humid districts of Asia and other continents are technically suitable for the rice production but still untreated or give the very low seed harvests because of salt and problem soils.The fifth part of irrigable soils in the world suffers from the unfavorable influence of high salinity of soil and from the problems of salination [1].The soil salination significantly limits the productivity of cultivated rice.In Russian Federation rice is cultivated on an area near 200 thousands of hectares.Near 80 thousands of hectares of the rice fields on South of Russia are incurred to salination to some extent [2].
The decrease of the crop capacity on the saline soils can be overcome by the improvement of the rice salt tolerance.That is why creation and introduction in agricultural production of the new productive sorts with the high tolerance to the salt stress is one of topical problems of this crop selection.
In last years was achieved the significant progress in elaboration of the molecular marking technologies and their use in the control of complicated agrotechnical signs by the marker selection [3].Technologies of molecular marking of the loci of salt tolerance allows select the plant forms with target genes and create sorts with required properties without using provocative backgrounds [4][5][6].The tolerance to salination at the different stages of growth is provided by the several independent genes and the main of them is Saltol placed in chromosome 1 [7].Its main function is the control of balance of Na + /K + ions in rice plants [8].The base of the breeding strategy for transfer of the concrete allele from the donor line to the recipient one is hybridization and use of molecular markers that allow control the transfer of genes to the posterity in every generation that increases the speed and reliability of the process of selection.

Aim of the work
Assessment of the rice hybrid forms F 2 -F 3 with the help of rolls and salt solution and also DNA-markers and PCR-analysis.Agricultural and Biological Scienсes

Materials and methods
There were used the sorts, collection samples and hybrids of Research Institute of grain crops of I. G. Kalinenko, Research institute of rice and agrogenetic institute (Hanoi, Vietnam).Hybrids of three combinations were received from the crossbreeding of salt tolerant donors IR 52713-2B-8-2B-1-2, IR 74099-3R-3-3 and NSIC Rc 106 with rareripe Krasnodar sort Novator.
The assessment of samples in the salt was carried out on the rolls of filtrated paper and polyethylene that were placed in the special trays then were added the salt solution in concentration 1,5 % NaCl [9].The control seeds were germinated on distilled water.Plants were grown in thermostat at temperature 26 °С, humidity 70 %.In 15 days was measured the length of roots and sprouts, the rate of seeds germination was calculated (Fig. 1).Fig. 1.The rice sprouts in 2 weeks on the salt solution (above) and on water (below) The rice plants were grown on checks of FSUE "Proletarskoe" in Rostov region.From the rice leaves selected on the breeding plots in laboratory conditions of the institute of biology SFI was separated the genomic DNA.At SSR analysis 2 pairs of primers chained with salt tolerance locus were used: RM493 and RM7075 [10].The statistical processing of experimental data was carried out using Excel program of Microsoft Office package.

Results of research
At the study of two Indian donors of the salt tolerance NSIC Rc 106 and FL 478 comparing with Krasnodar sort Novator and Rostov sorts Kuboyar, Svetly, Magnat, Boyarin, Yujanin and also with the collection sample of Chili 45-1-1 were established the significant differences in the length of roots and sprouts that varied on sorts and experiment variants (Table 1).
Among the studied sorts the maximal sprout length in the salt solution was in sorts Boyarin (7,33 cm) and Svetly (7,06 cm).The shortest sprouts were formed in sorts Novator (3,79 cm) and NSIC Rc 106 (3,91 cm).The most root length was noted in control in sorts Boyarin (10,57 cm) and Novator (10,52 cm), and in experiment -in Boyarin (5,24 cm), FL 478 (5,53) and NSIC Rc 106 (5,17).In this case two salt resistant samples demonstrated their advantage.The ratio of root length in experiment to the control one that characterizes the degree of tolerance turned out the most one just in them.In FL 478 this value is 89,2 %, in NSIC Rc 106-76,4 % (Fig. 2).

Fig. 2. Salt tolerance of the rice sorts, %
At the same time the tolerance to salination in Novator was minimal -42,2 %, little more in Kuboyar -46,3 %.In other samples the salt tolerance of roots was near 50 %.That is why FL 478 is most important for breeding.
Indian sorts were very late-maturing, blossomed in September and cannot mature in time in the field conditions of Rostov region.For transferring the tolerance genes to the native sorts was realized hybridization of donors NSIC Rc 106, IR 52713-2B-8-2B-1-2 and IR 74099-3R-3-3 with rareripe sort Novator [9].
From the plants of second generation were selected 90 rareripe well seeded samples (30 in each combination), that were analyzed by PCR method for the presence of introduced Saltol alleles.
In hybrid combination IR 52713-2B-8-2B-1-2×Novator were revealed two plants, homozygous on Saltol gene.17 plants were heterozygous, 9 -homozygous on recessive allele.Analogous results were received also on two other combinations of crossbreeding.Thus, in hybrid In general at studying genotypes on SSR-markers in each hybrid combination were revealed 2 forms homozygous on Saltol allele.They were seeded in the field on F 3 from which were selected the best forms for breeding and further study.
The split was far from the Mendel one 1:2:1.The number of heterozygotes was close to statistically probable one but the number of Saltol homozygotes turned up essentially lower than expecting one, and saltol -on the contrary, significantly higher.Thus, only 6 plants of 90 are homozygous on Saltol gene [10].
In 2015 the families of these homozygous and heterozygous forms were grown in the field where they essentially continued to split by many morphological signs and vegetative period.Among them were selected 340 best plants with heightened bushiness and seeding for receiving the sufficient amount of seeds for analysis and sowing.They were studied for salt tolerance by the roll method.The best lines are presented in rank order of the sprout length in the Table 2. Germination of seeds varied from 78 % (Novator) to 99 % (6675-1).In parent sort Novator the sprout length was 3,8 cm, and in hybrid samples -3,5-13,0 cm.Correspondingly, the root length in standard -4,4 cm, in samples -6,3-13,2 cm.The most strong, long seedlings were formed in lines 6907-1 (NSIC Rc 106×Novator), 7046-1 (IR 74099-3R-3-3×Novator) and 6975-1 (IR 52713-2B-8-2B-1-2×Novator) -up to 25,3 cm that is in 3 times more than in Novator.
They can be interesting for the marker rice selection for creation of the salt tolerant sorts.Agricultural and Biological Scienсes

Discussion of the results of research
The global change of climate to the warming leads to aridization and salination of soil causing the negative consequences for the production of rice that is the one of most important crops in the world.The climate change significantly worsens the unfavorable effects of abiotic stresses on the rice plants.The most part of rice lands is already affected by the increasing salinity level.But the problems of salt stress in plants can be effectively smoothed using the tolerant rice sorts.
In the result of previous studies we established the significant differences between the collection samples and sorts on the sprout and root length and separated 7 especially salt tolerant forms with the long sprouts and roots that must be recommended for breeding on tolerance to this stress-factor [11].
The earlier correlative analysis demonstrated that there is the mean positive correlation between the length of sprouts of the different rice sorts in the salt solution and salt tolerance r=0,63±0,16, between the length of roots and salt tolerance r=0,54±0,19 and strong positive correlation between the sprout and root length r=0,84±0,15 [12].
It is necessary to create the salt tolerant rice sorts using the donors of this sign at hybridization.But selection on the provocative backgrounds of the needed recombinants is rather complicated.That is why it is necessary to brood the rice sorts with the high potential of the crop capacity and seeds quality using the modern instruments of biotechnology [13,14].
But often it is difficult to transfer the salt tolerance genes in the high-yield sorts by the usual selection methods because of undesirable adhesion of genes that affect productivity and seed quality and occurs in hybrid posterity [15].We also observed the very low frequency of segregation from the hybrid population of homozygous forms with the dominant Saltol allelesnear 7 % instead of expected 25 %.It is connected with the fact that for analysis were selected the leaves from plants which seed is matured to the beginning of September, was beardless and did not fall.The sample could not be representative for the genetic analysis of split because the breeding selection was used for the hybrid populations.The single elite plants that carry Saltol found at PCR analysis appeared as a result of crossing over.Using this mechanism it is possible to separate the genes unfavorable for our conditions from the one of interest.In ideal, it would be better to transfer the one gene from the donor to the genetic base of commercial sort fit to the local conditions.It is possible with the help of backcrosses.
Last years the use of molecular selection allows create many rice sorts in different countries.In Vietnam with the use of МAS was improved the salt tolerance of widely cultivated rice sort OMCS2000 [16].In the different institutes is used the strategy of the reverse crossing with the marker control of transfer of favorable alleles of salination tolerance to the elite rice lines that significantly diminishes the terms of selection and make the laborious screening useful [17].
Because markers are chained with the certain signs of interest they can be used as biotechnological instrument for transferring the value genes of rice tolerance to the salt stress without undesirable adhesion [18].
Using backcrosses and marker control Vietnamese researchers successfully transferred the Saltol gene from the FL478 donor line to the BT7 sort that was selected as a recipient for the high quality of polished rice.Using SSR (RM493 and RM3412b) markers Saltol gene were successfully transferred to the genotype of BT7 that demonstrated the familiar phenotypic features comparing with original [19].They established that there is no essential difference on morphological signs between BT7 and separated lines with Saltol gene.
In our previous researches was established that in plants carrying the recessive and dominant Saltol alleles in homozygous state the height, length of panicle, the number of realized seeds, mass of seeds from panicle were almost at the same level.Analysis demonstrated that the plants with the salt tolerance gene have the development of quantitative signs at the level of unstable ones in the general growth conditions of Rostov region [20].
Their advantage in conditions of the real salination of soil can be revealed at the growing on the saline soils.It must be done after receiving homozygous lines F 6 -F 7 and breeding of their seeds in the number sufficient for the sowing on the large area.

Agricultural and Biological Scienсes
The limitation of our research is in the fact that the plants tolerance at the different stages of ontogenesis often differed.Tolerance to the salt stress at the germination stage not always coincides with such at the blossom stage and vice versa.That is why for more complete characteristic of sort it is necessary to study plants during their life using lysimeters, microchecks with saline soil or naturally saline fields.But the costs for such screening are significantly higher than the ones of laboratory methods.
Authors want to express gratitude to the FSBSI workers for the field works with our samples.Thank also to the workers of the Southern federal university for analysis of DNA-markers by their technical means.

Conclusions
1.With the help of rolls and salt solutions NaCl (1,5 %) were revealed the essential differences on the salt tolerance of the rice sorts and hybrids.
2. The split on Saltol gene differs from the theoretically expected one 1:2:1 because of selection of forms best on morphotype.
3. The marker selection allows separate the promising hybrids that can be interesting for creation of the salt tolerant rice sorts.

Table 1
Sizes of 15-days rice sprouts on the salt solution and water Agricultural and Biological ScienсesIR 74099-3R-3-3×Novator were detected 2 SS, 8 Ss and 14 ss; in hybrid NSIC Rc 106×Novator -2 SS, 13 Ss and 15 ss.Deviations in the results were affected by artificial selection of the rareripe plants best on morphotype.

Table 2
Germination and length of the sprouts of rice hybrids F 3 in rolls on 0,5 % NaCl solution in 15 days